La investigación que lleva a estos resultados ha recibido financiación de la Unión Europea del Séptimo Programa Marco (FP7/2007-2013) en virtud de acuerdo de subvención n ° 323267, de Electricité de France (EDF) y de l&#39;Agence Nationale de la Recherche - Santé-Environnement et Santé-Travail (ANR-SEST, Neurorad).

1. Los procedimientos con animales Este protocolo ha sido diseñado de acuerdo con la Directiva del Consejo Europeo de Comunidades de 24 de Noviembre de 1986 (86/609/CEE) y ha sido aprobado por nuestro comité institucional sobre bienestar animal (CETEA-CEA DSV IDF). <ol><li> Las inyecciones con edu / BrdU y la irradiación de E14.5 ratones preñados (Figura 2A). <ol><li> Preparar una solución de EdU a 1 mg / ml y de BrdU a 5 mg / ml en PBS. Realice una inyección intraperitoneal (IP) de 100 l de solución de EdU utilizando una aguja de 25 G en ratones embarazadas. 1.5 h más tarde, irradiar los ratones (irradiación corporal total) a 0 Gy o 2 Gy (0,6 Gy / min). Inmediatamente después, se inyectan (IP) 200 l de solución de BrdU utilizando una aguja de calibre 25 en los ratones embarazadas. </li></ol></li><li> Preparación de cortes coronales de cerebro embrionarias. <ol><li> En 1 hora o 4 horas después de la irradiación, la eutanasia a los ratones preñados por el dióxido de carbono. </li><li> Abra la cavi abdominalty más de tres centímetros con unas tijeras. Separar los dos cuernos uterinos del resto del útero mediante el seccionamiento de ambas extremidades de los cuernos. La transferencia de los cuernos uterinos en una placa de Petri que contenía PBS 0,6% de glucosa. Abra los cuernos uterinos con los fórceps con el fin de aislar los embriones. Retire la bolsa de líquido amniótico de cada embrión con unas pinzas. </li><li> Diseccionar cabezas de embriones con unas pinzas y fijarlos por inmersión en paraformaldehído al 4% (PFA, pH = 7,4) O / N a 4 ° C. </li><li> Enjuague cabezas de embriones en PBS durante al menos una noche a 4 ° C. Jefes se pueden mantener en PBS durante varios días. </li><li> Cabezas Insertar en parafina usando un procesador de infiltración al vacío como se indica en la Tabla 1. Jefes de incrustación también se puede realizar bajo una campana química para el etanol y baños de xilol, y luego en un horno de 65 ° C para los baños de parafina. </li><li> Preparar 5-m de espesor secciones coronales con un microtomo. </li><li> Montar en portaobjetos de microscopio recubiertos con polilisina. &lt;/ Li&gt; <li> Las diapositivas se pueden mantener a temperatura ambiente (TA) durante varios meses. </li></ol></li></ol> 2. Edu / BrdU tinción <ol><li> Desparafinización y antígeno desenmascarar <ol><li> Desparafinar las secciones del cerebro embebidos en parafina por inmersión de los portaobjetos en 3 baños de tolueno durante 5 min. </li><li> Se rehidrata durante 3 min en 2 baños de cada uno soluciones de la disminución de la concentración de etanol de la siguiente manera: etanol al 100%, etanol al 95%, etanol al 70%, y 5 min en H 2 O. Las diapositivas se pueden mantener en agua desionizada durante varias horas. </li><li> Hervir las rodajas durante 10 min en solución de citrato (10 mM, pH 6), y luego incubar en agua desionizada durante 5 min. </li></ol></li><li> Tinción EdU <ol><li> Realizar la detección de EdU de acuerdo con el protocolo del fabricante. Permeabilizar las células con 0,5% de Triton X-100 en PBS durante 10 min. </li><li> Preparar aditivo tampón 1X EdU diluyendo la solución 10 veces en agua desionizada. Esta solución debe ser recién hecho y utilizado en el mismo daY. </li><li> Preparar EdU cóctel de reacción, incluyendo EdU Alexa Fluor 488 azida. Es importante añadir los ingredientes siguiendo el orden indicado en la Tabla 2, de otro modo, la reacción no se procederá de manera óptima. Utilice el cóctel reacción EdU en los 15 minutos de preparación. </li><li> Retire la permeabilización de amortiguación (paso 2.1.1), luego se lava dos veces con PBS. </li><li> Añadir 150 l de EdU cóctel de reacción, poner un cubreobjetos, y se incuba durante 30 minutos en la oscuridad a temperatura ambiente. </li><li> Retire el cóctel reacción Edu, luego lavar una vez con PBS. </li></ol></li><li> Tinción BrdU <ol><li> Preparar tampón de saturación con 7,5% de suero de cabra y suero bovino fetal al 7,5% en PBS. Añadir 150 l de tampón de saturación en rodajas de cerebro, colocar un cubreobjetos, y se incuba durante 1 hora a TA para bloquear anticuerpo inespecífico vinculante. </li><li> Quite el cubreobjetos. Añadir 150 l de ratón anti-BrdU anticuerpo primario diluido a 1/300 en tampón de saturación, se puso un cubreobjetos y se incuban O /N a 4 ° C. </li><li> Quite el cubreobjetos, luego lavar 3 veces en PBS. </li><li> Añadir 150 l de anticuerpo de cabra anti-ratón-Alexa594 anticuerpo secundario diluido a 1/400 en tampón de saturación, poner un cubreobjetos, y se incuba durante 1 hora a TA. </li><li> Quite el cubreobjetos, luego lavar una vez con PBS. </li><li> Tinción nuclear: añadir 150 l de 4 &#39;,6-diamino-2-fenilindol (DAPI) a 1 g / ml en PBS, se puso un cubreobjetos y se incuba durante 2 minutos a temperatura ambiente. </li><li> Montar las diapositivas en medio de montaje. </li></ol></li></ol> 3. Análisis <ol><li> Examine las secciones del cerebro bajo un microscopio de fluorescencia con un objetivo 20X o un microscopio confocal y adquirir imágenes en tres canales (488 nm, 594 nm y UV) como separa archivos. </li><li> Pila de imágenes con el software Photoshop. </li><li> Analizar las secciones del cerebro en un sector estándar de la pared cerebral dorsomedial. Este sector es de 100 micras en su dimensión medial-lateral y está dividida en 18 contenedores (o más) de 10 μm de altura en su dimensión radial usando una cuadrícula superpuesta sobre imágenes (Figura 1) como se ha descrito previamente 31. <ol><li> Alinear la red, tales como el primer bin está en la superficie ventricular, con su eje largo paralelo a la frontera del ventrículo. Luego el número de la etiqueta EdU, BrdU y / o núcleos picnóticos (correspondiente a los núcleos apoptóticos) en cada bin. Número A núcleo situado en el límite de dos contenedores en el depósito que contiene su mayor parte, o en la papelera inferior, cuando el núcleo ocupa áreas iguales dentro de los dos contenedores. </li></ol></li><li> El análisis estadístico. </li></ol> Analizar al menos dos rebanadas corticales por embrión. Repita los experimentos en al menos 3 embriones de diferentes camadas. Los resultados deben ser dados como media ± error estándar de la media (SEM). Los análisis estadísticos se realizaron con Graphpad Prism usando ANOVA de dos vías y múltiples pruebas posthoc comparación Bonferroni.

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Los autores no tienen ningún conflicto de intereses.

El diseño experimental descrito aquí basado en la incorporación de EdU 1,5 horas antes de la irradiación y la incorporación de BrdU inmediatamente después de la irradiación permitió la demostración de que NSPCs son capaces de activar S y puntos de control G2 / M pero no el punto de control G1 / S durante la 1 ª hora después de un estrés genotóxico en el cerebro del ratón fetal. Se realizaron otros experimentos en los que se ha inyectado EdU en diferentes momentos después de la irradiación y Br...

Durante el desarrollo embrionario del cerebro, las neuronas de proyección de la corteza cerebral se generan en la zona ventricular, un epitelio pseudoestratificado compuesto de diferentes tipos de células madre neurales y células progenitoras (CPSN) que recubre los ventrículos laterales. Entre CPSN, las células gliales radiales (RGC), que sirven como células madre neurales, se someten a la migración nuclear interkinetic (INM): que realizan la mitosis en la superficie del ventrículo y la fase S en el límite basal de la zona ventricular (VZ) 1, 2,3. Ellos pueden dividir de forma simétrica para generar dos RGC o asimétricamente para generar una RGC y una neurona o una célula progenitora intermedia (IPC) 4, 5. IPC migrar a una capa superpuesta que proliferan llamada zona subventricular (SVZ), donde después de una última división simétrica generan dos neuronas de proyección inmaduros 6-8. Contrariamente a la RGC, IPC no sufren INM (revisado en 9). Neuronas recién generadas MIGRcomió radialmente a lo largo de las fibras radiales a través de la zona intermedia (IZ) para llegar a su destino final en la placa cortical (CP) 10, 8. La sincronización perfecta de todos estos eventos es esencial para un desarrollo cortical correcta. Por ejemplo, el interruptor de la proliferación a la diferenciación de las poblaciones progenitoras neuronales se controla por la duración de la fase G1 11, 12. El alargamiento de la fase G1 se correlaciona tanto con la diferenciación celular. Estrés genotóxico, como la radiación ionizante, el desarrollo del cerebro dañar seriamente (revisado en 13). Nosotros y otros han demostrado que NSPC son muy propensas a la apoptosis inducida por la radiación-14, 15,16. Las radiaciones ionizantes inducen ADN de doble filamento se rompe que son el daño más severo a las células proliferantes. Un componente esencial de la respuesta al daño del ADN (DDR) en el ciclo de las células es la activación de los puntos de control del ciclo celular en la transición G1 / S o G2 / M transición o durante Sfase (intra-S de control) 17-21. Ellos bloquean la progresión del ciclo celular para proporcionar tiempo para la reparación del daño en el ADN o eliminación de células demasiado dañados. En consecuencia, la muerte celular, así como la progresión del ciclo celular retardada pueden alterar el desarrollo del cerebro en respuesta a la exposición a radiaciones ionizantes 22-24. Por lo tanto, era interesante desarrollar un método para evaluar la activación de puntos de control del ciclo celular en CPSN en el cerebro de ratón embrionario irradiado. La progresión del ciclo celular es seguida de forma rutinaria utilizando la incorporación de un análogo de timidina, 5-bromo-2&#39;-desoxiuridina (BrdU). BrdU se incorpora durante la fase S del ciclo celular, cuando el ADN se replica. El uso de un anticuerpo contra BrdU a partir de entonces permite la detección de células que se encontraban en la fase S durante el pulso de BrdU. Un nuevo análogo de timidina, 5-etinil-2&#39;-desoxiuridina (edu) se detecta por una azida fluorescente. Las diferentes maneras de detectar Edu y B<html> RDU no reacciona de forma cruzada 25, lo que permite la detección simultánea de ambos análogos de timidina, que es útil para el estudio de la progresión del ciclo celular. Por lo general, las células se pulsaron con primera edu y pulsaron entonces con BrdU, en el que el tiempo entre las dos incorporaciones dura par de horas 25, 26. La adición de BrdU en medio de cultivo que contiene EdU preferentemente resulta en la incorporación de BrdU en el ADN con la exclusión de edu, mientras que la adición simultánea de EdU 25 con BrdU equimolar o medio equimolar a los resultados de los medios de comunicación en sólo incorporación de BrdU 27. Esto simplifica el protocolo de doble etiquetado mediante la eliminación de los pasos de lavado que normalmente se requieren para eliminar la primera etiqueta del medio de cultivo antes de la adición de la segunda etiqueta. Esto también es de especial interés para el estudio in vivo, donde los pasos de lavado no son posibles, para determinar el momento preciso de la entrada en la fase S o la salida de la población celular. t &quot;&gt; Recientemente, un método de etiquetado de doble pulso en el cerebro embrionario de ratón usando edu y BrdU 23, 24,22 se ha desarrollado para analizar la progresión del ciclo celular y el INM de CPSN después de la irradiación en el útero. Además, se ha demostrado que, durante fase S, células de ratón se replica en primer lugar las regiones de eucromatina y heterocromatina entonces el pericéntrica 28,29,30. Curiosamente, la heterocromatina pericéntrica de diferentes cromosomas agrupados en núcleos interfásicos para formar focos de heterocromatina también conocido como chromocenters y fácilmente detectable por tinción con DAPI como focos brillantes. Por lo tanto, los diferenciales Edu y BrdU tinciones de eucromatina y chromocenters nos ayudaron a analizar con mayor precisión la progresión de la fase S de NSPC. Este método permite la demostración de la aparente falta de G1 / S de control en NSCP 22-24, que es bastante sorprendente, ya que este punto de control se supone que es crítico para la estabilidad del genoma. Varios experimental diseños basados ​​en diversas combinaciones de edu y BrdU pulsos se han utilizado para analizar la progresión del ciclo celular en el telencéfalo ventral y dorsal. Aquí, le damos un ejemplo de protocolos que permiten el estudio del daño en el ADN aguda de NSPC dentro de las primeras 4 horas después de la irradiación en el útero de embriones E14.5 ratón.

<table border="0" cellpadding="0" cellspacing="0" width="727">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:228px;">EdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A10044</td>
			<td style="width:163px;">1 mg/ml</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">BrdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">B5002</td>
			<td style="width:163px;">5 mg/ml</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:228px;">IBL 637</td>
			<td style="width:212px;">CIS BIO International</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
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		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Tissu Tek VIP</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Microtome</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;">RM 2125 RT</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Triton X-100</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td align="right" style="width:125px;">93443</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:228px;">Click-iT EdU Alexa 488 imaging kit</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">C10083</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:228px;">Anti-BrdU</td>
			<td style="width:212px;">GE Healthcare</td>
			<td style="width:125px;">RPN202</td>
			<td style="width:163px;">1/300</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:228px;">Goat anti mouse-Alex594</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A11001</td>
			<td style="width:163px;">1/400</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Fluoromount</td>
			<td style="width:212px;">SouthernBiotech</td>
			<td style="width:125px;">0100-01</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Polysine slide</td>
			<td style="width:212px;">Thermo scientific</td>
			<td style="width:125px;">J2800AMNZ</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Paraformaldehyde</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">P6148</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:228px;">PBS</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">20012-068</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:228px;">DAPI</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">D9542</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Microscope BX51</td>
			<td style="width:212px;">Olympus</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Confocal microscope SPE</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Prism software</td>
			<td style="width:212px;">Graphpad</td>
			<td style="width:125px;"></td>
			<td style="width:163px;">Version 5.0c</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Photoshop software</td>
			<td style="width:212px;">Adobe</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
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assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress

Las neuronas de la corteza cerebral se generan durante el desarrollo cerebral de diferentes tipos de células madre y progenitoras neuronales (CPSN), que forman un epitelio pseudoestratificado que reviste los ventrículos laterales del cerebro embrionario. Tensiones genotóxicos, como la radiación ionizante, tienen efectos muy nocivos sobre el desarrollo del cerebro relacionada con la alta sensibilidad de NSPC. La elucidación de los mecanismos celulares y moleculares implicados depende de la caracterización de la respuesta al daño de ADN de estos tipos particulares de células, que requiere un método preciso para determinar CPSN progresión a través del ciclo celular en el tejido dañado. Aquí se muestra un método basado en sucesivas inyecciones intraperitoneales de Edu y BrdU en ratones embarazadas y aún más la detección de estos dos análogos de la timidina en secciones coronales del cerebro embrionario. Edu y BrdU se incorporan tanto en el ADN de las células de replicación durante la fase S y se detectan mediante dos técnicas diferentes (o azidaun anticuerpo específico, respectivamente), lo que facilita su detección simultánea. Edu y tinción de BrdU se determinan entonces para cada núcleo CPSN en función de su distancia desde el margen ventricular en una región estándar del telencéfalo dorsal. Por lo tanto esta técnica de doble etiquetado permite distinguir las células que progresaron a través del ciclo celular de los que han activado un punto de control del ciclo celular que conduce a la detención del ciclo celular en respuesta al daño del ADN. Un ejemplo de experimento se presenta, en el que se inyectó EdU antes de la irradiación y BrdU inmediatamente después y análisis realizados en el 4 horas después de la irradiación. Este protocolo proporciona un análisis preciso de la respuesta al daño de ADN aguda de CPSN en función de la fase del ciclo celular en la que se han irradiado. Este método es fácilmente extrapolable a muchos otros sistemas con el fin de determinar el impacto de un tratamiento particular en la progresión del ciclo celular en los tejidos vivos.

En el experimento descrito en la Figura 2, EdU se administró 1,5 horas antes de la irradiación y de BrdU sólo después de la irradiación. Cuatro tipos de células se distinguen en las rodajas corticales preparadas en 1 o 4 horas después de la irradiación, de acuerdo a la incorporación de cualquiera de EdU o BrdU, ambos o ninguno (Figuras 2A y 2B). Es importante destacar que, ni EdU ni la incorporación de BrdU cambiar el nivel de la apoptosis inducida por la radi...

Watch this Scientific Journal Video about Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress at JoVE.com

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress | Neuroscience | Video

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

La administración de dos análogos de timidina, edu y BrdU, en ratones embarazada permite el análisis de la progresión del ciclo celular en células progenitoras neurales y en el cerebro de ratón embrionario. Este método es útil para determinar los efectos del estrés genotóxico, incluyendo la radiación ionizante, durante el desarrollo del cerebro.

La evaluación de la progresión del ciclo celular de la madre neurales y células progenitoras en el cerebro en desarrollo del ratón después de estrés genotóxico

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

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15.2K vistas.  CEA DSV iRCM SCSR.  La administración de dos análogos de timidina, edu y BrdU, en ratones embarazada permite el análisis de la progresión del ciclo celular en células progenitoras neurales y en el cerebro de ratón embrionario. Este método es útil para determinar los efectos del estrés genotóxico, incluyendo la radiación ionizante, durante el desarrollo del cerebro. 

Video: Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse de imágenes en vivo de la relación clonal con células progenitoras neurales en el cerebro anterior El desarrollo de pez cebra

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Investigación

Neurociencias

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). ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Células vivas imágenes de células de los órganos sensoriales Precursor en Intacto Drosophila Las pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imágenes en vivo de la mitosis en el desarrollo de la corteza de embriones de ratón

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Derivación-Feeder libres de Células Progenitoras de la cresta neural Pluripotentes Células Madre

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Clasificación celular del Neuronales madre y progenitoras Las células del adulto Ratón zona subventricular y Live-imágenes de sus Dinámica del Ciclo Celular

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

Modelado de la infección del Virus Zika del desarrollo cerebro humano In Vitro usando células madre derivado Cerebral organitas

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.