This work was supported by the Brain Research Program through the National Research Foundation (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (Grants NRF-2015M3C7A1028790); and by a grant from the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (HI14C3347).

1. Preparación de Materiales y Medio de Cultivo <ol><li> Para la disección y la disociación de la SCZ, envolver la matriz de cerebro, hoja de doble filo de afeitar, y las pinzas con papel de aluminio y, a continuación, esterilizarlos en autoclave. </li><li> Preparar 50 ml de tampón de PBS fría para lavar todo el cerebro del ratón. </li><li> Establecer un microscopio de disección y preparar las herramientas quirúrgicas requeridas para la disección del cerebro (tijeras y pinzas en autoclave) y el aislamiento de la (jeringa de 1 ml, 30 aguja G, matriz de cerebro, y unas pinzas finas) SCZ. </li><li> Preparación de N2 Medio: <ol><li> Preparar F-12 / DMEM (+ L-glutamina, + bicarbonato de sodio) medio con 2% B27, 1% suplementos de N2, y 1% de penicilina-estreptomicina. Nota: El medio de crecimiento consiste en N2 medianas y factores de crecimiento (20 ng / ml de factor de crecimiento epidérmico purificada (EGF) y 20 ng / ml de factor de crecimiento de fibroblastos básico (bFGF2)). </li></ol></li><li> Preparar un tampón de digestión (40 unidades / ml de papaína, 2,4 unidad / ml dispase II, y 2% de penicilina-estreptomicina en PBS) para la digestión del tejido. </li><li> Preparación de la placa de revestimiento y Coverslip: <ol><li> Preparar poli-L-ornitina (OLP; 0,01%) y laminina (10 mg / ml disueltos en DH 2 O). Para revestir placas de 6 pocillos para el mantenimiento de SCZ-aNSCs como una monocapa o cubreobjetos de 18 mm para la inmunotinción, incubarlos con la OLP durante la noche a 4 ° C. Entonces ellos lavar 3 veces con DH 2 O. Dejar que las placas y los cubreobjetos se sequen después del último lavado. </li><li> A continuación, se incuban las placas con laminina durante la noche a 4 ° C. A continuación, lavar 3 veces con DH 2 O. Precaución: No secar la laminina, lo que afectará a la unión celular. Nota: Las soluciones de revestimiento se pueden reutilizar 3 veces. </li></ol></li></ol> 2. Aislamiento y disociación de la SCZ adulto <ol><li> Antes de la preparación de cultivo, colocar la matriz cerebro y la maquinilla de afeitar de doble filo en hielo. Nota: No congelar la matriz cerebral, debido a que el anchoain podría agregar a dicho informe. </li><li> Sacrificio un ratón (8 semanas de edad) por asfixia con CO2 o dislocación cervical. <ol><li> Cortar la cabeza con unas tijeras afiladas después de rociar 70% de etanol. Para inmovilizar la cabeza, sujete los dos lados de la cabeza con fuerza. Cortar la piel con unas tijeras en la línea media en dirección caudal rostral. Esto promueve la eliminación completa de la piel del cráneo. </li><li> Retire la parte caudal del cráneo (posterior a la lambda) en primer lugar y, a continuación, insertar la pinza entre el cráneo y el cerebro en la posición de la línea media dorsal. Agarrar el hueso parietal izquierda (o derecha) y cuidadosamente pele apagado. Repita este procedimiento para la eliminación del otro hueso parietal. Nota: La desconexión del nervio óptico y la eliminación de las meninges del cerebro que sea más fácil separar el cerebro del cráneo. </li><li> Transferir el cerebro a un tampón de PBS frío (25 ml) y enjuague dos veces para eliminar el exceso de sangre. </li></ol></li><li> Coloque el cerebro en la matriz cerebral en hielo y make cortes coronales para obtener rodajas de 1 mm de espesor. La transferencia de los cortes de cerebro (1 mM) que contenía las regiones SCZ que se encuentran en la parte posterior del cerebro a un PBS frío en una placa petri de plástico 35 mm. Precaución: Con el fin de obtener un plano paralelo de secciones coronales, asegúrese de que la fisura cerebro se coloca en la línea media de la matriz de cerebro; es crítico para evitar variaciones innecesarias en las secciones. Nota: Obtener cortes de cerebro a 2-3 mm posterior al bregma; esto permite la separación de la SCZ de la SVZ. </li><li> Bajo el microscopio de disección con un aumento bajo, micro-diseccionar el SCZ desde el área de materia blanca de la corteza y el hipocampo con un 6,9 aguja 30G doblada. A continuación, retire las regiones de la corteza por encima de la SCZ. Coloque la región SCZ disecados de las rebanadas en una placa petri de plástico de 35 mm en el hielo sin PBS frío. Nota: La contaminación de las regiones adicionales que contienen las neuronas maduras podría afectar a la viabilidad de aNSCs y la formación de neuroesferas bebido a que se someten a la muerte celular en condiciones de cultivo ANSC. </li><li> Usando una aguja doblada 30 G, cortar inmediatamente los tejidos disecados en trozos pequeños. Nota: Si se requiere más tiempo para diseccionar el tejido SCZ, sumerja los tejidos disecados en PBS frío antes de cortar. </li><li> Vuelva a suspender el tejido picado con 1 ml de tampón de digestión, la transferencia de tejido a un tubo de 15 ml que contiene 2 ml de tampón de digestión, y se incuba durante 30 min en un baño de agua a 37 °. Nota: Agitar el tubo cada 10 minutos para mezclar bien. </li></ol> 3. derivados de Zona subcallosal Cultura adulto Neural Stem Cell <ol><li> Toque suavemente el tubo para disociar el tejido digerido, y centrifugar el tubo a 145 g durante 5 min. Descartar el sobrenadante, se resuspendió el tejido digerido con 1 ml de medio precalentado N2 para lavar el tampón de digestión, y la pipeta suavemente la solución de muestra un máximo de 5 veces usando una pipeta P1000. Nota: El exceso de trituración con una estrechapunta de la pipeta puede disminuir la viabilidad celular y el crecimiento subsiguiente. </li><li> Centrifugar el tubo a 145 xg durante 5 min. Después de desechar el sobrenadante, volver a suspender el sedimento celular en 1 ml de medio N2. </li><li> Preparar 1 ml de medio N2 en una placa no recubierta 6 pocillos y añadir 1 ml de las células suspendidas para hacer un volumen final de 2 ml. </li><li> Añadir EGF (20 ng / ml) y bFGF (20 ng / ml) en cada pocillo. Agitar suavemente la placa de cultivo de 6 pocillos con la mano para mezclar los factores de crecimiento añadidos a las células sembradas. Mantenga la placa de 6 pocillos en un incubador de CO2 al 37 ° C y 5%. </li><li> Añadir EGF (20 ng / ml) y bFGF (20 ng / ml) a cada pocillo cada día durante 8 días. Cada tercera día, añadir 200 l de los medios de comunicación N2 para mantener el volumen aproximado 2 ml del medio. </li></ol> 4. pases de células madre neurales como neuroesferas y para los cultivos en monocapa <ol><li> Reunir las neuroesferas y transferirlos a un nuevo tubo cónico de 15 ml. Nota: El número de neuroesferas (&gt; 50 micras de diámetro) per bien desde el SCZ de un solo cerebro de ratón era de 64,3 ± 7,31, que era menor que la de la SVZ (190,5 ± 6,33) 9. </li><li> Incubar las neuroesferas con 0,5 ml de tampón de disociación de 5 minutos en un baño de agua a 37 ° para disociar las neuroesferas en células individuales. neuroesferas primarias pueden disociarse en células individuales y se mantuvieron durante varios pasajes como los cultivos de neuroesferas o monocapa. Nota: La expansión de SCZ-aNSCs como una monocapa es superior a neuroesferas porque SCZ-aNSCs pueden ser pasados&gt; 10 veces en un formato de cultivo en monocapa, pero &lt;5 veces en un formato de la cultura neurosphere. Precaución: SCZ-aNSCs exhiben una fuerte agregación, y es difícil de disociar en células individuales. Por lo tanto, el formato de la cultura neurosphere no se recomienda para la expansión de células de rutina. </li><li> pipetear suavemente la solución de la muestra de arriba abajo con una pipeta P1000 menos de 5 veces y se centrifuga el tubo a 145 g durante 5 min. Eliminar el sobrenadante y volver a supasar las neuroesferas con 1 ml de medio N2. Nota: Durante trituración con una pipeta de punta estrecha puede disminuir la viabilidad celular y el crecimiento subsiguiente. </li><li> Para contar las células, hacer una mezcla 1: 1 de la suspensión celular (10 l) y la solución de azul de tripano al 0,4%, y luego contar el número de células vivas en un hematocitómetro. Después de recubrir una placa de 6 pocillos con PLO / laminina, la placa de las células a 2,5 x 10 5 células / ml con 2 ml de medio de N2 para cada pocillo. Nota: Los cambios en la densidad celular pueden afectar su condición y potencial de diferenciación. </li><li> Mantener los SCZ-aNSCs con un tratamiento diario de factores de crecimiento (2 ml, 20 ng / ml) durante 5 días, y los pasaje ellos. </li></ol> 5. Diferenciación de los derivados de Zona subcallosal células madre adultas Neuronales <ol><li> ANSCs placa sobre un cubreobjetos de 18 mm recubiertos de laminina PLO / con 1 x 10 5 células / ml en 1 ml N2 con factores de crecimiento (20 ng / ml) para la diferenciación de los SCZ-aNSCs. </li><li> El día siguiente,cuando las células están firmemente unidos al cubreobjetos, intercambiar el medio de crecimiento con N2 para eliminar los factores de crecimiento. </li><li> Después de 6 días, se lavan las células diferenciadas con 1 ml de PBS para eliminar los restos celulares y fijarlos para inmunotinción. Nota: BrdU se puede incorporar en el ADN recién sintetizadas de las células proliferantes. Por lo tanto, si se desea, BrdU (10 mg / ml) puede ser añadido a las células vivas antes de la fijación de las células. </li></ol> 6. Las células madre neurales adultas inmunotinción y diferenciada Progenitores <ol><li> Para la inmunotinción, se lavan las células con PBS y fijarlas con 4% PFA durante 20 min a temperatura ambiente. Precaución: PFA es altamente tóxico; evitar el contacto con la piel y los ojos. </li><li> Retire el 4% PFA, lavar las células fijadas con PBS 3 veces, y luego almacenarlos a 4 ° Cuntil que son necesarios para la inmunotinción. </li><li> Se incuban las células en cubreobjetos con solución de bloqueo (3% de albúmina de suero bovino y 0,1% de Triton X-100 en 1 x PBS) durante al menos 30 min a temperatura ambiente. </li><li> Preparar los anticuerpos primarios en solución de bloqueo fresco y se incuban las muestras durante la noche a 4 ° C. <ol><li> La inmunotinción los aNSCs <ol><li> Tinción de las aNSCs utilizando anticuerpos anti-BrdU anti-nestina y. Antes de la fijación, añadir 20 g de BrdU a las aNSCs y incubarlos durante 2 h. Realizar la etapa de desnaturalización usando HCl 2 N durante 20 minutos a 37 ° CAntes la realización de la etapa de bloqueo. </li></ol></li><li> La inmunotinción los progenitores diferenciadas: <ol><li> Utilizar anticuerpos anti-glial fibrilar ácida de la proteína (GFA) anti-O4, anti-bIII-tubulina, y para etiquetar los progenitores diferenciadas. </li></ol></li></ol></li><li> Lavar las muestras con PBS 3 veces y se incuban con anticuerpos secundarios conjugados con colorantes fluorescentes (1: 500) en solución de bloqueo durante 30 min a temperatura ambiente. A continuación, lavar las muestras 3 veces con PBS. Nota: Use anticuerpos secundarios que responden a los anfitriones de la primariaanticuerpos. Hoechest33343 (1: 2000) se utiliza para la tinción nuclear. </li><li> Añadir la solución de montaje a un portaobjetos de vidrio y proceder con el montaje. Observe y la imagen de muestra en un microscopio confocal en múltiples longitudes de onda: FITC (488 nm), Cy3 (543 nm), Cy5 (647 nm), y Hoechest33343 (405 nm). </li></ol>

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The authors declare no conflicts of interest.

Este documento describe un protocolo detallado para generar células madre neurales de la SCZ ratón adulto y mantenerlas para diversas aplicaciones. Hay tres pasos críticos para el establecimiento del sistema de cultivo in vitro necesaria para purificar y expandir SCZ-NSC. En primer lugar, es importante asegurarse de que la región SCZ se diseca precisamente hacia fuera de otras regiones neurogénicas potenciales (Figura 1B). Secciones gruesas y precisos que contienen las regiones SCZ se obtu...

NSCs have characteristics of self-renewal and multiple-lineage differentiation. To confirm these properties, a neurosphere culture system has widely been used. The neurosphere culture system was developed in the early 1990s and served as a standard stem cell culture system1. Depending on self-renewal potency, NSCs continuously proliferate and generate a cell mass in a suspension culture. The number of cells and the size of the neurosphere are considered to be closely associated with the proliferation properties of the NSCs. Monolayer cultures are also widely used for the maintenance and differentiation of NSCs. Compared to the neurosphere culture, the monolayer culture system provides better homogenous maintenance and expansion of NSCs2. These two well-developed culture systems have contributed to the characterization of aNSCs in vitro. 
NSCs reside in different brain regions, such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus3-5. The subcallosal zone (SCZ) of the caudal subcortical white matter is recognized as a novel neurogenic region6-8. It was recently reported that the SCZ-aNSCs have therapeutic potential in traumatic brain injury9. Compared to other neurogenic regions, the SCZ resides along the subcortical white matter. In the human brain, subcortical white matter occupies a larger region than in the mouse brain10. Therefore, an understanding of the characteristics of SCZ-aNSCs using an in vitro culture system is important in order to promote the potential use of these cells for neural regeneration. Precise dissection of the desired brain region is required to rule out possible contamination by unwanted regions containing active or quiescent NSCs. For instance, aNSCs in non-neurogenic regions can be activated and produce new neural cells in injured brains or during in vitro culturing11. To obtain NSCs from the SCZ, cells were collected from brain slices containing the SCZ. Then, a careful micro-dissection of the SCZ region was performed using a fine needle. To generate the neurosphere from the SCZ, micro-dissected SCZ tissue chunks were dissociated into single cells and then cultured as a suspension in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After SCZ-aNSCs form neurospheres, they also can be maintained as neurospheres or monolayers for expansion. This procedure also demonstrates immunostaining processes with various markers for the detection of NSCs and their progeny after their expansion and differentiation in a monolayer culture. Here, a visual protocol of the SCZ-aNSC culture system is presented. This protocol contains detailed instructions for the micro-dissection of the SCZ region and for the maintenance and passaging of the cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>+L-glutamin, +Sodium bicarbonate</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>BIOSOLUTION</td>
			<td>BP007a</td>
			<td>1X dilution</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociattion buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>04-942-078-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>3126</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ICT</td>
			<td>AT-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>WPI</td>
			<td>555229F</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Storz</td>
			<td>E3321-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain matrix (1mm)</td>
			<td>RWD</td>
			<td>68707</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-edged razor</td>
			<td>DORCO</td>
			<td>ST-300</td>
			<td></td>
		</tr>
		<tr>
			<td>30 gage needle</td>
			<td>SUNGSHIM</td>
			<td>N1300</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>SPL</td>
			<td>10035</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>100mm dish</td>
			<td>SPL</td>
			<td>10090</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>Cover slip (18mm)</td>
			<td>Deckglaser</td>
			<td>111580</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well dish</td>
			<td>SPL</td>
			<td>32012</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>6 well dish</td>
			<td>SPL</td>
			<td>32006</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>Coating materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLO</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td>0.01%</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Primary antibodies&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore</td>
			<td>MAB353</td>
			<td>mouse (1:1000)</td>
		</tr>
		<tr>
			<td>EGFR</td>
			<td>Abcam</td>
			<td>ab2430</td>
			<td>rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>DCX</td>
			<td>Santa Cruz</td>
			<td>SC8066</td>
			<td>goat (1:500)</td>
		</tr>
		<tr>
			<td>Tuj1</td>
			<td>Sigma</td>
			<td>T2200</td>
			<td>rabbit (1:2000)</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Invitrogen</td>
			<td>13-0300</td>
			<td>rat (1:1000)</td>
		</tr>
		<tr>
			<td>O4</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td>mouse (1:500)</td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Abcam</td>
			<td>ab6326</td>
			<td>Rat (1:500)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td rowspan="12">1:500</td>
		</tr>
		<tr>
			<td>anti-mouse cy3</td>
			<td>Jackson</td>
			<td>715-165-151</td>
		</tr>
		<tr>
			<td>anti-mouse 647</td>
			<td>Jackson</td>
			<td>715-606-150</td>
		</tr>
		<tr>
			<td>anti-rabbit 488</td>
			<td>Alexa</td>
			<td>A21206</td>
		</tr>
		<tr>
			<td>anti-rabbit cy3</td>
			<td>Jackson</td>
			<td>711-165-152</td>
		</tr>
		<tr>
			<td>anti-rabbit 647</td>
			<td>Jackson</td>
			<td>711-605-152</td>
		</tr>
		<tr>
			<td>anti-goat 488</td>
			<td>Alexa</td>
			<td>A11055</td>
		</tr>
		<tr>
			<td>anti-goat cy3</td>
			<td>Jackson</td>
			<td>705-165-147</td>
		</tr>
		<tr>
			<td>anti-goat 647</td>
			<td>Invitrogen</td>
			<td>A21447</td>
		</tr>
		<tr>
			<td>anti-rat 488</td>
			<td>Invitrogen</td>
			<td>A21208</td>
		</tr>
		<tr>
			<td>anti-rat cy3</td>
			<td>Jackson</td>
			<td>712-166-150</td>
		</tr>
		<tr>
			<td>anti-rat 647</td>
			<td>Jackson</td>
			<td>712-605-153</td>
		</tr>
		<tr>
			<td>Immunostaining materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Millipore</td>
			<td>82-100-6</td>
			<td>3% BSA with 0.1% Triton X-100 in PBS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>usb</td>
			<td>22686</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>Biosesang</td>
			<td>P2031</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechest33342</td>
			<td>Life Technology</td>
			<td>H3570</td>
			<td>Dye for staining nuclei</td>
		</tr>
	</tbody>
</table>

isolation and culture of adult neural stem cells from the mouse subcallosal zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Definir el sistema de cultivo de aNSCs de la región neurogénica desconocido es esencial para la comprensión de estas células y para el desarrollo de su potencial uso en la reparación del cerebro 12. Se sabe que NSCs en diferentes etapas de desarrollo o en diferentes regiones comportan de manera diferente 3,4. Recientemente, se informó de que las células derivadas-SCZ exhiben potenciales diferenciales para la diferenciación neuronal in vivo e in ...

Watch this Scientific Journal Video about Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone at JoVE.com

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Aislamiento y cultivo de células madre adultas neural de la Zona subcallosal Ratón

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

isolation-culture-adult-neural-stem-cells-from-mouse-subcallosal

Research

JoVE Journal

Neuroscience

15.0K vistas.  Korea University College of Medicine. Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described. 

Video: Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

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 ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Aislamiento y cultivo de células troncales de la cresta neural de los folículos del cabello humano

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

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 ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Cultivo de células madre neurales de Regiones convencionales y no convencionales del cerebro adulto de roedores

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Aislamiento y cultivo de células endoteliales del cerebro anterior embrionarias

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Un ratón, dos culturas: Aislamiento y cultivo de adultos Neural las células madre de las dos zonas neurogénicos de ratones individuales

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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. ...

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.