Este trabajo fue apoyado por fondos de la Fundación Overstreet.

1. Elementos que deben estar preparados antes de proceder a la chapa del celular: <ol><li> Volumen apropiado de medio completo NSC se prepara mezclando NeuroCult NSC medio basal y NeuroCult Suplementos NSC proliferación en una proporción de 9:1, respectivamente. </li><li> Una alícuota de NeuroCult NCFC medio libre de suero sin citoquinas se descongela. </li><li> El medio es calentado en un baño de agua a 37 ° C. </li><li> Disponibilidad de soluciones de factor de crecimiento epidérmico (EGF), factor de crecimiento de fibroblastos (b-FGF) en la concentración de 10 mg / ml y heparina en el 0,2% son preparados con anticipación. </li><li> Dependiendo del tamaño del experimento, varios platos de 35 mm de cultivo de tejidos son necesarios para la placa de las células y un 150-200cm placa Petri de plástico también es necesaria para mantener los platos de 35 mm y un duplicado tercer plato de 35 mm de agua. </li></ol> 2. Preparación de células: Dependiendo de su experimento, las células pueden ser preparados a partir de una fuente de adultas o embrionarias (tejido primario disociados o neuroesferas disociadas). Diseccionar los tejidos de adultos / embriones de ratón del sistema nervioso central (SNC) o disociar neuroesferas adulto / embriones derivados de 1,2 como se describió anteriormente y luego: <ol><li> La suspensión de células individuales se hace pasar por un filtro de malla de 40 micras de tamaño con el fin de expulsar a los no disociados grupos. </li><li> 10μl de la suspensión celular se mezcla con 90μl de azul tripán para llevar a cabo un recuento de células. Nota: Otros diluciones adecuadas de células también puede ser utilizado. </li><li> Si se utiliza embrionarias o adultas derivadas primaria las células nerviosas, diluir la suspensión celular a una concentración de 6,5 x 10 5 células / ml en medio completo NSC. Si utiliza las células de neuroesferas disociadas derivados de las células neuronales adultas o embrionarias, se diluye la suspensión celular a una concentración de 2,2 x 10 5 células / ml en medio completo NSC. </li></ol> 3. Las células siembra en semi-sólida N-CFCA Medio: <ol><li> El volumen adecuado de los siguientes componentes se mezcla con el fin, según el número de repeticiones. Aquí se mezcla la cantidad necesaria para dos réplicas o duplicados platos. Para los números de repeticiones adicionales, por favor consulte la tabla 1. <ul><li> 1,7 ml de NeuroCult NCFC medio libre de suero sin citoquinas </li><li> 330 L de NeuroCult NSC Suplementos proliferación </li><li> 6,6 l de EGF (10μg/mL) </li><li> 32 l de penicilina / estreptomicina </li><li> 3,3 L de b-FGF (10μg/mL) sólo es necesario si el cultivo de células derivadas de células adultas neural. </li><li> 3,3 l de heparina (0,2%) sólo es necesario si el cultivo de células derivadas de células adultas neural. </li><li> 25μL de suspensión celular (de 2,2 x 10 5 células / ml de células de neuroesferas disociadas o 6,5 x 10 5 células / ml células primarias de tejido del SNC disociado). Nota: El número final de células plateado debe ser alrededor de 2500 células por 35 mm placa de cultivo para neuroesfera células derivadas y 7500 células por placa de cultivo de 35 mm de células primarias. En algunos casos, la densidad celular revestimiento final tiene que ser ajustado mediante la realización de un experimento de valoración de la célula. Para el análisis estadístico, el número total de colonias detectadas tras 21 días de la cultura debe estar dentro del rango de al menos 50 a 150 colonias. </li></ul></li><li> El medio que contiene las células se mezcla suavemente y luego 1,3 ml de la solución de colágeno frío se transfiere a la suspensión celular y se mezcla bien con la pipeta suave para evitar la introducción de las burbujas. </li><li> La solución mezclada se imparte en el centro de cada plato de cultivo de 35 mm (alrededor de 1,5 ml / placa) y los platos se inclinan suavemente con un movimiento circular para que la mezcla uniformemente a lo largo de la superficie del plato. . </li><li> El duplicado placas de 35 mm de la cultura se colocan en una placa de Petri de 100 mm. La tapa del nuevo plato de 35 mm se elimina y el plato abierto también se coloca en el mismo 100 mm placa de Petri. Agua estéril se añade a este plato mm abrir 35 para mantener la humedad óptima durante el período de incubación. Placas cuadradas bioensayo (245 mm) se utilizan cuando se replican más placas de 35 mm se han establecido. Una vez más, son 2 o 3 abrir placas de 35 mm que contiene agua estéril. </li><li> Las placas se trasladan a una incubadora a 37 ° C, 5% CO2 y 95% de humedad. El colágeno se congela por aumento de la temperatura y la formación de gel se producirá dentro de aproximadamente una hora. Las culturas no debe ser alterada durante este tiempo. </li><li> Cultivos de células se incuban durante 21 días (las diferencias en tamaño de la colonia se pueden distinguir claramente después de 21 días). </li></ol> 4. Preparación de medio de refuerzo y de alimentación de la Cultura: Como la N-CFCA cultivos se incuban durante un período prolongado de tiempo (21 días), los cultivos deben ser alimentados con la NeuroCult completa adecuada medio reposición recién preparada de la siguiente manera: <ul><li> 4,5 ml de medio NeuroCult NSC basal se mezcla con 0,5 mL de NeuroCult NSC Suplementos proliferación y los factores de crecimiento se añade: </li><li> 250 L de 10μg/mL EGF (para dar una concentración final de 0,5 mg / ml FEAG) </li><li> 125 L de 10μg/mL b-FGF (para dar una concentración final de 0.25μg/mL b-FGF) sólo es necesario si el cultivo de células derivadas de células adultas neural. </li><li> 125 l de 0,2% con heparina, sólo es necesario si el cultivo de células derivadas de células adultas neural. </li><li> 60 L de la adecuada reposición NeuroCult medio completo (para que las células embrionarias o adultas), se añade en el centro de cada plato de ensayo NCFC una vez cada 7 días durante toda la incubación NCFC cultura de ensayo (21 días). </li></ul> 5. Anotando N-CFC colonias Ensayo derivados y calcular la frecuencia de bona fide NSCs y células progenitoras neurales: <ul><li> Cada plato de 35 mm cultura se coloca en un plato de puntuación cuadriculada con el tamaño de la malla de 2,0 mm x 2,0 mm y luego los dos platos se colocan en la platina del microscopio. </li><li> El uso de baja potencia (2,5 veces - 5 veces) de objetivo, cada plato se escanea y las colonias se califican en función de su tamaño. </li><li> Las colonias se clasifican en dos categorías principales: <ol><li> Menos de 2 mm de diámetro </li><li> ≥ 2 mm de diámetro </li></ol></li></ul> 6. Los resultados representativos: Como en el ensayo neuroesfera, las células se sembraron en la N-CFC ensayo comienzan a proliferar y hacer pequeñas colonias de células dentro de 3 a 7 días después de placas (Figura 1). Dentro de dos semanas, las colonias de diferentes tamaños se pueden distinguir. Mientras que la mayoría de las colonias tienden a dejar de crecer después de 14 días, algunas colonias siguen aumentando de tamaño. El día 21, las colonias se pueden clasificar en una de las cuatro categorías: 1) menos de 0,5 mm de diámetro, 2) 0.5 a 1 mm de diámetro, 3) 1 - 2 mm de diámetro y 4) ≥ 2 mm de diámetro. En la práctica, las colonias más pequeñas que el diámetro de 2 mm se les conoce como progenitoras derivadas (Figura 2) y las colonias ≥ 2 mm de diámetro se conoce como NSC derivados (Figura 3). El número de colonias ≥ 2 mm de diámetro por las células chapada total, representa la frecuencia real de la buena fe, las células madre neurales a largo plazo la auto-renovación y capacidad de múltiples posibilidades. El total de la formación de neuroesfera frecuencia en una población de células en particular después de 7-8 días en un experimento paralelo NSA ha estimado que es similar a la colonia de la formación total de la frecuencia de la misma población celular después de 21 días en un experimento de N-CFCA. La N-CFCA sin embargo, constituye un requisito más permisivas a fin de cada celda puede mostrar su potencial proliferativo completo. <table border="1"><tbody><tr><td> Componente </td><td> 2 repeticiones </td><td> 3 repeticiones </td><td> 4 repeticiones </td></tr><tr><td> NeuroCult NCFC medio libre de suero sin citoquinas </td><td> 1700 </td><td> 2550 </td><td> 3400 </td></tr><tr><td> NeuroCult NSC proliferación Suplementos </td><td> 330 </td><td> 495 </td><td> 660 </td></tr><tr><td> EGF (10 mg / mL) </td><td> 6.6 </td><td> 9.9 </td><td> 13.2 </td></tr><tr><td> bFGF (10 mg / mL), sólo para las células adultas </td><td> 3.3 </td><td> 4.95 </td><td> 6.6 </td></tr><tr><td> Solución de heparina (0,2%), sólo para las células adultas </td><td> 3.3 </td><td> 4.95 </td><td> 6.6 </td></tr><tr><td> Penicilina / estreptomicina (1:100) </td><td> 32 </td><td> 48 </td><td> 64 </td></tr><tr><td> Las células en: 2,2 x 10 5 células en cultivo / ml o 6,5 x 10 5 células primarias / mL </td><td> 25 </td><td> 37.5 </td><td> 50 </td></tr><tr><td> Solución de colágeno </td><td> 1300 </td><td> 1950 </td><td> 2600 </td></tr></tbody></table> Tabla 1. Componentes de la cultura completa del ensayo N-CFC. <img src="/files/ftp_upload/2639/2639fig1.jpg" alt="Figura 1" /> Figura 1. Colonias Representante de la N-CFCA cultura de NSCs paso del ratón E14 7 días después de la siembra. Las colonias pueden mostrar diferente morfología y tamaño. Original de la ampliación; 4x <img src="/files/ftp_upload/2639/2639fig2.jpg" alt="Figura 2" /> Figura 2. Representante colonias progenitoras derivadas de diferentes tamaños en la N-CFCA cultura de paso un E14 NSCs ratón 21 días después de la siembra. Como se ve, las colonias tienen una morfología diferente, pero todos están menos 2 mm de tamaño. Original de la ampliación; 4x <img src="/files/ftp_upload/2639/2639fig3.jpg" alt="Figura 3" /> Representante de la figura 3. Celular de buena fe, madre derivadas de colonias en la N-CFCA cultura de paso un E14 NSCs ratón 21 días después de la siembra. Colonias de células madre derivadas puede mostrar morfología diferente (verel video), pero todos son ≥ 2 mm de diámetro. Original de la ampliación; 4x

<ol>
	<li>Azari, H., Rahaman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J Vis Exp. , (2010).</li><li>Azari, H., Sharififar, S., Rahaman, M., Ansari, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J Vis Exp. , (2011).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Reynolds, B. A., Rietze, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+Neural+Stem+and+Progenitor+Cells+in+the+Neural+Colony+Forming+Cell+Assay.">Enumeration of Neural Stem and Progenitor Cells in the Neural Colony Forming Cell Assay.</a> Stem Cells. , (2008).</li><li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26, 979-987 (2008).</li><li>Louis, S. A., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+and+Neural+Colony-Forming+Cell+Assays.">Neurosphere and Neural Colony-Forming Cell Assays.</a> Protocols for Neural Cell Culture. 10, 1-28 (2010).</li></ol>

Uno de los autores, Luis A. Sharon, está afiliada a StemCell Technologies Inc, fabricante de algunos reactivos utilizados en este estudio.

A pesar de ensayo neuroesfera 3,1,2 es el método más común para aislar y expandir las células madre neurales de una variedad de fuentes como el tejido del SNC adultas y embrionarias, que no puede medir con precisión la frecuencia de NSC en una población mixta de células precursoras neuronales (tallo y progenitores), ya que no es una relación uno a uno entre el número de neuroesferas y el número de bona fide células madre 4. Para hacer frente a esta limitación, el original NSA ha sido adaptado para permitir la madre neuronales y progenitores para alcanzar su plena capacidad de proliferación durante tres semanas 5-7. A diferencia de los líquidos de la NSA, en la N-CFCA las colonias son en realidad clones derivados, como la matriz de colágeno semi-sólida que previene la migración de las células de una sola placa y la agregación de las colonias. Para obtener resultados consistentes con esta prueba se recomienda: <ol><li> Garantizar una única suspensión de células en la suspensión celular original. Pase su única suspensión de células a través de un filtro de malla de 40 micras de tamaño con el fin de expulsar a los no disociados grupos. </li><li> Mantenga la solución de colágeno en el hielo o en la 4 ° C nevera. El colágeno es el último elemento que se añade a la mezcla de la suspensión celular, ya que se congela por el aumento de la temperatura. </li><li> No te olvides de alimentar a la cultura de cada semana. Añadiendo el medio que contiene la reposición del factor de crecimiento (s) una vez cada 7 días permitirá que las células que siguen proliferando en el período de cultivo extendido. </li><li> La densidad celular final de placas han sido optimizados para las células derivadas de cualquiera de las células neuronales primarias o cultivadas de modo que el número total de colonias detectadas tras 21 días de la cultura en el NCFC-A está dentro del rango de al menos 50 a 200 colonias. Esta gama permite la detección de las pocas colonias NSC derivados&gt; 2 mm de diámetro en las muestras de tiempo que proporciona un número suficiente de colonias en los análisis estadísticos. Dependiendo de la fuente de células de partida, un número significativamente menor (&lt;50) y un mayor número de colonias (&gt; 250) a veces se obtienen. Colonias muy pocos se traducirá en las colonias&gt; 2 mm de diámetro que debajo de los niveles de detección, mientras que las colonias también se traducirá en muchos anddepletion hacinamiento de los componentes del medio de cultivo que resulta en un recuento de colonias inexacta. Por lo tanto la densidad de recubrimiento celular tendrá que ser ajustado en consecuencia (es decir, aumentando o disminuyendo el número total de células sembradas) </li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>NeuroCult NSC Basal Medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05700</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>STEMCELL</td>
<td>05701</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NCFC medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05720</td>
<td>&nbsp;</td>
<tr>
<td>0.05% 	trypsin-EDTA</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25300-062</td>
<td>&nbsp;</td>
<tr>
<td>Soybean trypsin inhibitor </td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6522</td>
<td>&nbsp;</td>
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<td>Cell strainer </td>
<td>Sieve</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
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<td>Pen/Strep </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
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<td>T25 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>136196</td>
<td>&nbsp;</td>
<tr>
<td>T80 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>178905</td>
<td>&nbsp;</td>
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<td>Collagen</td>
<td>Reagent </td>
<td>STEMCELL</td>
<td>04902</td>
<td>&nbsp;</td>
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<td>EGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>2028-EG</td>
<td>&nbsp;</td>
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<td>b-FGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>3139-FB</td>
<td>&nbsp;</td>
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<td>Heparin</td>
<td>Growth factor</td>
<td>Sigma</td>
<td>H4784</td>
<td>Reconstituted in PBS</td>
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<tr>
<td>15 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
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<td>50 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352070</td>
<td>&nbsp;</td>
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<td>35 mm culture dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27100</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27500</td>
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neural-colony forming cell assay: an assay to discriminate bona fide neural stem cells from neural progenitor cells

El ensayo neuroesfera (NSA) es uno de los métodos más utilizados para aislar, expandir y también calcular la frecuencia de las células madre neurales (NSC). Además, este sistema de cultivo libre de suero también se ha empleado para expandir las células madre y determinar su frecuencia a partir de una variedad de tumores y tejidos normales. Se ha demostrado recientemente que una relación de uno a uno, no existe entre la formación y neuroesfera NSCs. Esto sugiere que la NSA como se aplica actualmente, sobreestima la frecuencia de los comités directivos en una población mixta de células precursoras neurales aisladas, tanto del cerebro de los mamíferos embrionarias y adultas. Este vídeo demuestra un colágeno prácticamente novela basada semi-sólido ensayo, la neuro-formadoras de colonias ensayo de células (N-ACCP), que tiene la capacidad de discriminar se derivan de las células progenitoras en base a su potencial a largo plazo de proliferación, y por lo tanto proporciona un método para enumerar frecuencia NSC. En la N-CFCA, colonias ≥ 2 mm de diámetro se derivan de las células que cumplen todos los criterios funcionales de un Consejo de Seguridad Nacional, mientras que las colonias &lt;2 mm se derivan de los progenitores. El procedimiento de N-CFCA se puede utilizar para las células preparadas a partir de diferentes fuentes, incluyendo adultos primaria y cultivadas, las células embrionarias de ratón del SNC. Aquí se utilizan las células preparadas a partir de un pasaje neuroesferas generadas a partir del día 14 embrionario del cerebro para llevar a cabo los ratones N-CFCA. Los cultivos se repone con el medio de la proliferación de cada siete días durante tres semanas para permitir que las células chapada a exhibir su potencial proliferativo completo y luego la frecuencia de las progenitoras neurales y de buena fe las células madre neurales se calcula, respectivamente, contando el número de colonias que son &lt;2 mm y los que son ≥ 2 mm en referencia al número de células que se cultivaron inicialmente.

Watch this Scientific Journal Video about Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells at JoVE.com

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

Este protocolo de vídeo se muestra cómo se discrimina y enumerar bona fide las células madre neurales en una población mixta de células precursoras neurales con el neuronales formadoras de colonias de células de ensayo.

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). ...

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Research

JoVE Journal

Neuroscience

22.0K vistas.  University of Florida. Este protocolo de vídeo se muestra cómo se discrimina y enumerar bona fide las células madre neurales en una población mixta de células precursoras neurales con el neuronales formadoras de colonias de células de ensayo.

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

El aislamiento y la expansión de las células troncales nerviosas de ratones adultos mediante el ensayo de neuroesfera

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

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 ...

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 ...

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. ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Enumeración de células madre neurales Usando ensayos clonales

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

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.

Desarrollo de un protocolo de refinado para Trans-escleral transplante Subretinal de células epiteliales del pigmento retiniano humano a ojos de rata

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identificación de marcadores de superficie celular de células madre neuronales primarias y células progenitoras por etiquetado metabólico de sialoglicano

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...