Damos las gracias a Denis Flipo consejo de procesamiento de imágenes y análisis, y David W. Silversides en cuyo laboratorio se generó la línea de ratón Gata4p [5 kb]-GFP. La investigación en el laboratorio Pilon es financiado por CIHR, NSERC, FRQS y FRQNT.

Declaración de Ética Los experimentos con ratones se realizaron siguiendo Consejo Canadiense de Cuidado de Animales Directrices para el cuidado y la manipulación de los animales utilizados en la investigación médica. Protocolos que implican la manipulación de los animales fueron aprobados por el comité de ética institucional de la Universidad de Quebec en Montreal (Comité de Protección Institutionnel des Animaux; número de referencia 0512-R3-650-0513). 1. Preparación de geles de colágeno Trabajar de forma estéril, bajo una campana de cultivo de tejidos. <ol><li> Preparar completa 5x DMEM (medio esencial modificado de Eagle) incluyendo antibióticos estándar. Disolver 3,37 g de polvo de DMEM y 0,925 g de NaHCO3 en 20 ml de agua. Esterilizar mediante el paso a través de un filtro de 0,22 micras. Añadir 2,5 ml de penicilina estéril 100x / estreptomicina y 25 ml de suero bovino fetal inactivado por calor estéril. Almacenar a 4 ° C. </li><li> En el hielo, mezclar 800 lde la solución de colágeno I (3,77 mg / ml en 0,02 N de ácido acético, esterilizada por filtración), 600 l de DMEM completo 5x y 17 l de 1 N de NaOH. Se diluye con agua estéril a un volumen final de 3 ml. Incluya factores de crecimiento apropiados en la mezcla. Utilizamos típicamente GDNF a 10 ng / ml para estimular la migración NCC entérico. </li><li> Depósito de unos 480 l en cada pocillo de una sola fila de una placa de 24 pocillos. Evite las burbujas. Las filas restantes se pueden utilizar para probar el efecto de otros factores de crecimiento sobre la migración celular. </li><li> Deje que el colágeno se polimeriza al menos 1 hora en un incubador estéril a 37 ° C. </li></ol> 2. Disección de animales 16 <ol><li> Configure los apareamientos y compruebe si hay tapones vaginales a la mañana siguiente. E0.5 Día siendo el mediodía del día en que el tapón vaginal se encuentra, aislar a la hembra y esperar 12 días hasta E12.5. </li><li> Anestesie ratones preñados con isoflurano y la eutanasia por inhalación de CO2. </li><li> Rocíe el ratón con etanol al 70%. Levante las s abdomenparientes y abrir la cavidad abdominal con tijeras de disección. </li><li> Extirpar el útero en una placa de Petri de vidrio lleno de helado de PBS (solución salina tamponada con fosfato). Cortar el útero transversalmente entre hinchazones deciduum individuales para aislar cada sitio de la implantación del embrión. </li><li> El trabajo en cada lugar de la implantación por separado en otra placa Petri de vidrio lleno de helado de PBS. Bajo un microscopio de disección, utilizar unas pinzas finas para eliminar la capa muscular del útero. </li><li> Abra el saco vitelino visceral y amnios para revelar el embrión. Tener cuidado al cortar los vasos sanguíneos de unirse al embrión a la placenta / visceral del saco vitelino, a medida que se entrelazan con los intestinos en desarrollo. </li><li> Cortar la cabeza del embrión en el cuello. </li><li> Inserte un fórceps cerrados en la cavidad abdominal de un embrión, justo por encima del hígado y de color rojo oscuro y dejar que el fórceps abierto de sí mismo (dejar de aplicar presión) para hacer una abertura transversal en la cavidad abdominal. Saque las pinzas abiertas hacia abajoel extremo posterior del embrión para abrir el abdomen completamente. </li><li> Coge el tejido conectivo detrás del hígado y tire de las tripas del abdomen, con cuidado de no romper el intestino (colon se une al ano). </li><li> Cortar el colon para liberar las agallas del resto del embrión. El corte puede hacerse en cualquier lugar a lo largo del colon. Reserve la parte de cola para más adelante. </li><li> Desentrañar el tejido conectivo desde el ciego, a continuación, el resto de los intestinos. Tenga cuidado de no herir los intestinos mientras lo hace. </li><li> Cortar el hígado y el estómago (en el extremo rostral del intestino delgado), así como los mesonefros y crestas genitales si algunos son presente. </li><li> Aislar el intestino delgado. Una vez más, tenga cuidado de no cortar el intestino. A partir de ahora, la orientación rosrrocaudal del tejido intestinal se puede seguir utilizando la fuerte curvatura presente en el extremo rostral. Deje el intestino delgado en PBS a temperatura ambiente durante un tiempo tan corto como sea posible antes de emRopa de cama (vea el paso 3.3). </li><li> Por último, anote el número de somitas cola para cada embrión para determinar con precisión la etapa de desarrollo embrionario. </li></ol> 3. La sección del embrionarias Intestinos <ol><li> Antes de proceder con disecciones de embriones, se funden 1,5 g de agarosa en 100 ml de PBS (solución salina tamponada con fosfato) y mantenga a 50 ° C. </li><li> Vierta agarosa derretida en un molde de inclusión (por ejemplo, un tubo de microcentrífuga cerrado 2 ml que ha sido cortado a lo largo de extirpar aproximadamente 1/4 de la pared del tubo). Deje que la agarosa se enfríe a aproximadamente 42-45 ° C, debe ser sólo ligeramente caliente al tacto. </li><li> Incrustar el intestino embrionario justo antes de que se solidifique agarosa (esto se puede evaluar con fórceps consejos y 36-38 ° C ocurrir alrededor). Sosteniendo el intestino con fórceps por el extremo rostral plegada, tire de ella muy lentamente a través de la agarosa a lo largo de la longitud del molde. Esto ayuda a mantener el intestino recto, mientras que los conjuntos de agarosa. Suelte el temitir tan pronto como comienza a resistirse a ser movido. No pierda de vista la orientación rostral-caudal del intestino. </li><li> Ponga el molde en la nevera 2-3 minutos, para asegurar que la agarosa se ha establecido por completo. </li><li> Saque la agarosa del molde (deslizándolo fuera del tubo Eppendorf abierto). Con una hoja, sacar el exceso de agarosa en ambos extremos, haciendo cortes perpendiculares al intestino. </li><li> Pegue el extremo rostral del intestino / agarosa bloquear abajo en la escena del metal de un microtomo con cuchilla vibrante. Recorte el exceso de agarosa de los lados del bloque delgado / agarosa. </li><li> Montar el escenario de metal en la cámara de microtomo vibrante. Si es necesario, ajuste el ángulo del escenario para que el intestino es tan vertical como sea posible (por lo tanto perpendicular a la hoja). Cubra la muestra con helado de PBS. Traiga la hoja microtomo hasta unos pocos milímetros bajo la superficie de amortiguación. </li><li> Hacer 200 micras vibratome transversales cortes de intestino-caudal más pequeño, lo que garantiza que el correoach rodaja de agarosa contiene una sección intestinal completa. </li></ol> 4. Cultura de intestinal explantes <ol><li> Depositar suavemente el rodajas delgado / agarosa recién cortadas plana sobre los geles de colágeno con pinzas, colocar una rebanada a la mitad de cada bien. </li><li> Incubar 3 días a 37 ° C, en una atmósfera húmeda al 5% de CO 2, para permitir la migración de NCC fuera del explante. </li><li> Tomar las rodajas delgado / agarosa el gel de colágeno muy suavemente con fórceps. Tenga cuidado de no dañar el gel a continuación. </li><li> Evitar tocar el gel de colágeno directamente durante la incubación y de lavado de los pasos subsiguientes con el fin de garantizar que el patrón de la migración de células no sea perturbada. Fijar con 500 l de 4% de PFA (paraformaldehído) (en PBS) por pocillo 1 hora a temperatura ambiente. </li><li> Reemplazar el fijador con 500 l de DAPI (4 &#39;,6-diamino-2-fenilindol) solución (5 mg / ml en PBS) por pocillo e incubar 10 min a temperatura ambiente. </li><li> Lavar cada pocillo 3xcon 500 l de PBS durante 5 min. </li><li> Fotografía las células fluorescentes (canales GFP y DAPI) incrustados en el gel de colágeno dentro de cada pocillo. </li></ol> 5. Análisis de Imagen Hemos hecho un amplio uso de ImageJ 17 para procesar y cuantificar las imágenes generadas después del cultivo de explante. <ol><li> Comience por imágenes de un micrómetro de diapositivas con la misma ampliación que las fotografías de células fluorescentes. Medir la longitud de una micra en píxeles (con la función Straight Line). </li><li> Establecer la escala (Analizar / Set Escala; número de entrada de píxeles / micras). </li><li> Para cada fotografía la fluorescencia de GFP, cambiar el formato de la imagen a escala de grises de 8 bits (Image/Type/8-bit). </li><li> Ajusta la intensidad de la señal (Imagen / Ajustar / Brillo / Contraste). </li><li> Reste el ruido de fondo si es necesario (Proceso / Restar fondo, ajustar el radio de la esfera rodante). </li><li> Establecer un umbral para resaltar las celdas y los grupos de células (Imagen / Ajustar / Umbral), lan aplicar una cuenca para dividir a los grupos (Proceso / Binary / Cuenca). </li><li> Analizar las partículas para especificar las regiones de interés (ROI) y generar estadísticas de número de células (Analizar / Analizar Partículas; mínimo el tamaño del conjunto de excluir píxeles restantes). </li><li> Establecer opciones de medición para incluir el diámetro de Feret (Analizar / Medidas Set). </li><li> Grupo ROI y medir en su conjunto para determinar el diámetro de la Feret, una indicación de la propagación de células (Analizar / Herramientas / ROI Gerente / Más / O, entonces Director de ROI / Agregar y ROI Gerente / Measure). </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+developmental+disorders+of+the+enteric+nervous+system.">Development and developmental disorders of the enteric nervous system.</a> Nat. Rev. Gastroenterol. Hepatol. 10 (1), 43-57 (2012).</li><li>Sasselli, V., Pachnis, V., Bursn, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enteric+nervous+system.">The enteric nervous system.</a> Dev. Biol. 366 (1), 64-73 (2012).</li><li>Sanchez, M. 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D., 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=Neurotrophin-3+is+required+for+the+survival-differentiation+of+subsets+of+developing+enteric+neurons.">Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.</a> J. Neurosci. 21 (15), 5620-5636 (2001).</li><li>Goldstein, A. M., Brewer, K. C., Doyle, A. M., Nagy, N., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+necessary+for+neural+crest+cell+migration+and+ganglion+formation+in+the+enteric+nervous+system.">BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system.</a> Mech. Dev. 122 (6), 821-833 (2005).</li><li>Jiang, Y., Liu, M. T., Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+and+DCC+in+the+guidance+of+migrating+neural+crest-derived+cells+in+the+developing+bowel+and+pancreas.">Netrins and DCC in the guidance of migrating neural crest-derived cells in the developing bowel and pancreas.</a> Dev. Biol. 258 (2), 364-384 (2003).</li><li>Ramalho-Santos, M., Melton, D. A., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signals+regulate+multiple+aspects+of+gastrointestinal+development.">Hedgehog signals regulate multiple aspects of gastrointestinal development.</a> Development. 127 (12), 2763-2772 (2000).</li><li>Natarajan, D., Marcos-Gutierrez, C., Pachnis, V., de Graaf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+signaling+by+receptor+tyrosine+kinase+RET+for+the+directed+migration+of+enteric+nervous+system+progenitor+cells+during+mammalian+embryogenesis.">Requirement of signaling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis.</a> Development. 129 (22), 5151-5160 (2002).</li><li>Young, H. M., Hearn, C. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+pre-+and+post-gastrulation+expression+of+Gata4+within+cells+of+the+inner+cell+mass+and+migratory+neural+crest+cells.">Novel pre- and post-gastrulation expression of Gata4 within cells of the inner cell mass and migratory neural crest cells.</a> Dev. Dyn. 237 (4), 1133-1143 (2008).</li><li>Nagy, N., Goldstein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin-3+regulates+neural+crest+cell+proliferation+and+differentiation+in+the+hindgut+enteric+nervous+system.">Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system.</a> Dev. Biol. 293 (1), 203-217 (2006).</li><li>Nagy, A., Gertsenstein, M., Vintersen, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: a laboratory manual. , 209-250 (2003).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Byth, K. F., Thomas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AZD5438,+a+potent+oral+inhibitor+of+cyclin-dependent+kinases+1,+2,+and+9,+leads+to+pharmacodynamic+changes+and+potent+antitumor+effects+in+human+tumor+xenografts.">AZD5438, a potent oral inhibitor of cyclin-dependent kinases 1, 2, and 9, leads to pharmacodynamic changes and potent antitumor effects in human tumor xenografts.</a> Mol. Cancer Ther. 8 (7), 1856-1866 (2009).</li></ol>

Los autores no tienen nada que revelar.

Mostramos cómo nuestro vivo técnica de cultivo ex explante se puede utilizar para cuantificar con precisión entérica potencial de migración NCC en presencia de GDNF. Dicha cuantificación exacta es facilitada en gran medida por el uso de 200 micras de espesor secciones intestinales vibratome lugar de grandes piezas de tamaño aproximado, como se describe anteriormente 11,12,15. De hecho, esto nos permite trabajar con un número razonable de las células en un entorno altamente reproduci...

Células de la cresta neural (CCN) son un tipo de célula transitoria única de vertebrados que forma muchos derivados durante el desarrollo embrionario. Surge Esta población de células en la frontera de la placa neural, adyacente al ectodermo no neurales 1. Durante la neurulación, la flexión de los lugares placa neural NCC lo largo del borde dorsal del tubo neural formando. NCC luego se someten a una transición epitelio-mesenquimal, segregar y migrar lejos del tubo neural. NCC colonizar diversas estructuras embrionarias, incluyendo el tracto digestivo donde forman todo el sistema nervioso entérico (ENS), una red interconectada de los ganglios nerviosos incrustado en la pared intestinal. Tan recientemente revisado 2,3, muchos genes han sido implicados en el desarrollo de esta intrincada estructura. La mayor parte de la ENS se deriva de un pequeño grupo de NCC procedente del tubo neural vagal (es decir, en torno al límite prospectivo posterior del cerebro / médula espinal) 4.Estos progenitores neurales llegan al intestino anterior alrededor del día embrionario (e) 9.0 en ratones y luego migran caudalmente en el mesénquima intestinal hasta aproximadamente e15.0 colonizar todo el intestino embrionarias. Un subconjunto menor de progenitores neurales de colon también es proporcionada por sacra NCC, que invaden el intestino posterior en la dirección opuesta hasta el ciego 4. Tanto vagal y sacral NCC requiere múltiples migración, proliferación, supervivencia y diferenciación de las señales promotoras para asegurar la formación completa de la ENS. En este sentido, los modelos animales - especialmente ratones genéticamente modificados - han sido fundamentales para la identificación de varios ligandos extracelulares esenciales: GDNF (factor neurotrófico derivado de células gliales), la endotelina-3, la neurotrofina-3, (proteínas morfogenéticas óseas) BMP, Netrina , así como Sonic y erizo indio (Shh y Ihh) 5-10. De éstos, el GDNF señalización a través de la RET receptor transmembrana tirosina quinasa (Reordenación durante la transfección) se reconoce como the vía más importante para la atracción y la orientación de NCC y dentro del intestino embrionario. GDNF es secretada por el mesénquima intestino y forma un gradiente de rosrrocaudal espaciotemporalmente controlado que es directamente quimioatrayentes para entérico NCC, que expresan RET 11,12. Entre otras funciones, la ENS regula el movimiento en el tracto digestivo a través de su interacción con el músculo liso de la pared intestinal. Ausencia de los ganglios de los nervios en la región terminal de los resultados del intestino en la enfermedad de Hirschsprung: contracción tónica del segmento afectado conduce a la obstrucción, la acumulación de aguas arriba del material digerido y distensión masiva del intestino y el abdomen. La enfermedad de Hirschsprung ocurre aproximadamente uno de cada 5.000 nacidos vivos. Se cree que el patrón de migración rostro-caudal del entérica NCC siendo el principal factor que contribuye a la etiología de la enfermedad de Hirschsprung. El colon, más alejado de la fuente de la migración de NCC y última porción del bowel ser colonizado, es más susceptible a defectos en la formación ENS. De acuerdo con su papel crucial en la migración NCC entérica, la alteración de la señalización de GDNF / RET es la principal causa genética conocida de la enfermedad de Hirschsprung 13. Para un mejor estudio de NCC y desarrollo ENS, hemos generado una línea de ratones transgénicos - llamado Gata4p [5 kb]-GFP 14 - en la que NCC migratoria están etiquetados con la proteína verde fluorescente (GFP). A continuación perfeccionado un ensayo ex vivo la migración celular, adaptado a partir de los trabajos publicados por otros grupos 11,12,15, que ahora permite la cuantificación precisa de entérica potencial de migración NCC en la presencia de varios factores de crecimiento, tales como el GDNF.

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

Células de la cresta neural (CCN) son una población de células multipotentes transitoria y que se origina en el tubo neural dorsal y migra extensamente por todo el embrión de los vertebrados en desarrollo. Además de proporcionar la glía y neuronas periféricas, NCC generar los melanocitos, así como la mayor parte del esqueleto cráneo-facial. NCC la migración y la diferenciación está controlada por una combinación de su origen axial a lo largo del tubo neural y su exposición a las señales extracelulares regionalmente distintas. Tal contribución de ligandos extracelulares es especialmente evidente durante la formación del sistema nervioso entérico (ENS), una compleja red interconectada de los ganglios nerviosos que controla localmente (entre otras cosas) el movimiento del músculo intestinal y la motilidad intestinal. La mayor parte de la ENS se deriva de un pequeño grupo inicial de NCC que emprenda un largo viaje con el fin de colonizar - en una rostral caudal a la moda - la longitud completa del intestino prospectivo. Entre varias vías de señalización que se sabe queinfluir en la colonización entérica NCC, GDNF señalización / RET es reconocido como el más importante. De hecho, la secreción espaciotemporalmente controlada del ligando RET GDNF por el mesénquima intestinal es el principal responsable de la atracción y la orientación de RET-expresando entérica NCC hacia y dentro del intestino embrionario. Aquí, se describe un ensayo ex vivo la migración celular, haciendo uso de una línea de ratón transgénico que posee la etiqueta fluorescente NCC, que permite la cuantificación precisa de entérica potencial de migración NCC en la presencia de varios factores de crecimiento, incluyendo el GDNF.

Los siguientes resultados son representativos de lo que se puede obtener con la técnica descrita aquí (Figura 1). El uso de factores de crecimiento (es decir, GDNF) estimula la migración de expresión de GFP-entérica NCC fuera del explante intestinal y en el gel de colágeno (Figura 2). Aunque algunas células salen del explante en ausencia de factores de crecimiento, éstos en su mayoría no son GFP-etiquetados y representan la entrada pasiva. Es necesario quitar la rebana...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Se presenta un ensayo in vivo la migración ex célula que permite la cuantificación precisa de los nervios potencial de migración de células de la cresta entérica en la presencia de varios factores de crecimiento.

Un ensayo de migración celular cuantitativa para murinos entéricas Neuronales Progenitores

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

9.2K vistas.  UQAM.  Se presenta un ensayo in vivo la migración ex célula que permite la cuantificación precisa de los nervios potencial de migración de células de la cresta entérica en la presencia de varios factores de crecimiento. 

Video: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Una delgada del cráneo ventana Técnica para la crónica de dos fotones En vivo Imágenes de la microglía murina en los modelos de la neuroinflamación

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Investigación

Neurociencias

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

El establecimiento de embriones de ratón con células madre neurales Cultura mediante el ensayo de neuroesfera

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural formadoras de colonias ensayo de células: un ensayo para discriminar Bona Fide células troncales nerviosas de células progenitoras neurales

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

El análisis de la migración de células troncales neurales Crest utilizando una versión modificada del ensayo Zigmond Cámara

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Ratón reproducible aplastamiento del nervio ciático y posterior evaluación de la regeneración mediante el análisis de músculo entero Monte

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Análisis de la migración de la cresta neural y la diferenciación de las especies cruzadas Trasplante

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

El ensayo neuroblasto: un ensayo para la generación y enriquecimiento de las células progenitoras neuronales de la diferenciación de la progenie de células madre neurales Usando citometría de flujo

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Aislamiento y cultivo de células de la cresta neural de embriones murinos del Tubo Neural

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

Un ensayo Galvanotaxis por análisis de la cinética precursoras neurales migración celular en un campo aplicado externamente directo Corriente Eléctrica

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.