Este estudio fue apoyado por subvenciones del Vanderbilt-Ingram Cancer Center de Apoyo Grant (P30 CA068485), la Fundación cerebrales infantiles del tumor y de los Institutos Nacionales de Salud (NS042205).

1. Microdisección de portadores de tumores del cerebelo, la disociación del tejido del tumor y de las planchas <ol><li> Recuperación del tejido tumoral <ol><li> Meduloblastoma enfermos ratones portadores se runted a menudo, muestran hidrocefalia y típicos síntomas neurológicos, incluyendo parálisis posterior y el fracaso para recuperar la posición en caso de vuelco. Para recuperar el tejido tumoral, la eutanasia a los ratones por inhalación de dióxido de carbono. Es importante no realizar la dislocación cervical, un procedimiento que genera una presión en el cráneo y posterior pueden poner en peligro la integridad del tejido tumoral. </li><li> La decapitación se realiza inmediatamente después de la muerte con un par de tijeras, quitar el pelo y el tejido muscular tanto como sea posible para una buena visualización del cráneo. Limpie la superficie del cráneo con Kimwipe empapado con etanol al 95%. </li><li> Use tijeras finas para cortar una abertura a lo largo de la línea media del cráneo, y eliminar el tejido del cráneo con unas pinzas finas, momento en el que todo el cerebro incluyendo el cerebelo con tumores está expuesto. </li><li> Mientras que el cerebelo de los adultos sanos pantalla bien definida hemisferios y el vermis, el cerebelo de ratones portadores de tumores a menudo son ampliadas, amorfa con una superficie lisa y los vasos sanguíneos visibles. El uso de técnicas estériles, recuperar el tumor cerebeloso con unas pinzas y colocar en helado de PBS sin Mg 2 + y Ca 2 +. </li></ol></li> Nota: todos los instrumentos se esterilizan en etanol al 95% antes de su uso. <li> La disociación del tejido tumoral <ol><li> Transferir el tejido tumoral de PBS al 50% Accutase (diluido en PBS), que es de aproximadamente 4 veces el volumen del tejido del tumor, el picadillo de tejido con tijeras finas durante 3 minutos a temperatura ambiente, seguido de incubación a 37 ° C durante 4 minutos , tras lo cual el tejido se somete a pipetear repetitivo con un Pipetman de 1 ml durante 3 minutos. Este método se debe dar una mezcla de células individuales y pequeños agregados celulares. </li><li> Diluir la suspensión celular 3 veces con PBS y centrifugar durante 5 minutos a 1000 g para precipitar las células. Resuspender el botón celular en medio fresco cultura neural de células madre y la placa en un gelatinizada 60 mm Primaria plato de cultivo de tejidos. Utilizamos platos Primaria para la fijación de placas mejoradas primera pasajes posteriores pueden ser sembradas en platos de cultivo de tejido normal. </li></ol></li></ol> Nota: platos Escudo cultura con 0,1% de gelatina durante al menos 30 minutos. Prepare frescos de células madre neurales medio de cultivo sobre el medio Neurobasal con la glutamina, la pluma Strep-, N2, B27, EGF humano (25 ng / mL) y el FGF básico (25 ng / mL). 2. Enriquecimiento, mantenimiento y expansión de las células del tumor meduloblastoma por pases seriados Por lo general, obtener muchas colonias después de 1 semana de la siembra inicial (Figura 1). Estas colonias pueden ser disociados y replated en nuevos platos gelatinizada para el enriquecimiento de la población de células tumorales. Primer cambio de medio de cultivo nuevo, basta con utilizar un Pipetman de 1 ml para separar mecánicamente las colonias seguido por pipeta repetitiva durante 4 minutos para obtener una suspensión celular (no Accutase es necesario). Compruebe bajo un microscopio para asegurarse de que grupos de células grandes se han disociado. Entonces prescindir de la suspensión de células en nuevos platos gelatinizada para el cultivo de más y pasar por el mismo procedimiento para pasajes adicionales. Las células tumorales sin ataduras, las células sanguíneas y otros tipos de células son eliminadas por serie re-chapados, dejando a las células tumorales unido a una rápida expansión. Cambio medio de cultivo en el segundo día después de la siembra inicial, y cada dos días después. 3. La tinción de inmunofluorescencia y el examen de las células cultivadas 1. Crecen las células tumorales en cubreobjetos de vidrio El día 1, un lugar cubreobjetos de vidrio en cada uno de una placa de 6 pocillos, a continuación, agregar 0,1% de gelatina durante 30 minutos a 37 ° C. Semillas de aproximadamente 2-4X 10 5 células tumorales por ml. Las células deben adjuntar toda la noche y el medio de células madre neurales cambiado el día 3. Tinción se lleva a cabo el día 4. 2 tinción de inmunofluorescencia Retire el medio de cultivo y las células se lavan dos veces con helado de PBS. Fijar las células con el recién hechas el 4% PFA durante 15 minutos. En pocas palabras lavar las células dos veces con PBS y permeablize con 0,3% Triton (en PBS) durante 5 minutos. En pocas palabras lavar las células dos veces con PBS y se bloquea con suero de cabra al 10% durante 40 minutos. Se incuban las células con el anticuerpo primario durante 90 minutos, luego lavar las células tres veces con PBS, durante 5 minutos cada uno. Se incuban las células con el anticuerpo secundario durante 60 minutos, luego lavar tres veces con PBS, 5 minutos cada uno. Monte cubreobjetos en diapositivas con 5 ml de un medio de montaje acuoso y realizar microscopía confocal. 4. La diferenciación de las células tumorales Quitar medio de células madre neurales y brevemente lavar las células con PBS dos veces. Añadir medio de diferenciación en el día 1, el cambio medio en el día 4 y determinar el nivel de diferenciación por inmunofluorescencia en el día 7. El medio de diferenciación consiste en DMEM, Pen Strep-y el 10% de suero fetal bovino. 5. Resultados representante Resultados de la morfología Después del tratamiento Accutase y pipeta repetitiva suave, el tejido del tumor debe ser en su mayoría se disocian en pequeños agregados celulares y células individuales. Muchas colonias considerable se puede observar a los 5 días después de placas inicial, como se muestra en la Figura 1. Las células tumorales de proliferación a menudo son bi-polar con una alta tasa núcleo / citoplasma. Estas células se ven normalmente irradia de pequeños agregados celulares adheridos a la superficie gelatinizada. Células de la sangre, pequeño y redondo, generalmente se retiran los medios de comunicación por los cambios posteriores y revestimientos de serie. Después de varios pasajes, se puede observar uniformemente distribuidos, de proliferación Ki67 + en las células tumorales y poca contaminación con otros tipos de células (Figura 2). Expresión de marcadores de células madre neuronales, análisis clonal y diferenciación en múltiples linajes Para determinar si las células de proliferación derivado de disociar las características del tumor del cerebelo muestra de tejido de células madre tumorales, se realizó una caracterización adicional por la expresión de marcadores de células madre, el análisis clonal y la potencia de diferenciación. Hemos encontrado que estas células tumorales aisladas altamente expresar marcadores de células madre neurales, como Sox2 y Nestin (Figura 3). Como se recuperaron los tejidos de SmoM2-YFP tumor, todas las células tumorales expresan YFP. De acuerdo con un estudio reciente informa que los cilios primarios son importantes para el desarrollo de Shh vía dependiente de meduloblastoma 12, SmoM2-YFP expresión fue localizado con claridad a los cilios de las células tumorales Sox2 + (Figura 3). Cuando se siembran a una densidad clonal de 300 células por ml de medio de cultivo, se observó la expansión clonal de una célula tumoral única a una colonia importante (Figura 4). Además, estas células fueron capaces de diferenciarse en diversos tipos de células neuronales y gliales cuando se cultiva en condiciones de diferenciación a favor de (Figura 5). <img src="/files/ftp_upload/2086/2086fig1.jpg" alt="Figura 1" /> Figura 1. Morfología de una colonia de proliferación después de la siembra de primera de tejido meduloblastoma disociados. Por lo general, se forma una colonia importante dentro de 5 días después de la siembra inicial de tejido tumoral disociado, y un representante se ilustra aquí, en campo claro. Bi-polar, células alargadas irradian de un agregado de núcleo de célula denso. Unido a las células tumorales son células pequeñas, redondas rojas de la sangre, que el agotamiento gradual de recubrimientos de serie. <img src="/files/ftp_upload/2086/2086fig2.jpg" alt="Figura 2" /> Figura 2. Morfología de las células de meduloblastoma establecido más allá de los tres pasajes. Esta imagen de campo claro se muestra el aspecto típico de las células de meduloblastoma establecido después de varios pasajes. Las células permanecen en su mayoría bi-polar de alta nuclear / citoplasma. Nos muestran una tinción tubulina acetilada de las células tumorales, la mayoría de los cuales son el ciclismo como lo demuestra una fuerte expresión de Ki67. <img src="/files/ftp_upload/2086/2086fig3.jpg" alt="Figura 3" /> Figura 3. Establecido células de meduloblastoma expresan marcadores de células madre neurales. SmoM2-YFP marca las células que expresan constitutivamente activa Smo, por lo tanto, actividad de la vía de Shh. Todas las células de las líneas celulares establecidas meduloblastoma expresó YFP, y la mayoría de ellos co-expresaron altos niveles de varios marcadores de células madre neurales, como Nestin y Sox2. Curiosamente, cuando se realizó la detección YFP señal con la potencia del láser durante un mínimo de microscopía confocal, se detectó la señal concentrada YFP en los cilios de las células Sox2 +. Esta observación es consistente con un reciente informe de 12 que se describe el papel esencial de los cilios en el desarrollo de Shh vía dependiente de meduloblastoma. <img src="/files/ftp_upload/2086/2086fig4.jpg" alt="Figura 4" /> Figura 4. Estableció las células tumorales experimentar expansión clonal. Después de la disociación en suspensión de células individuales, las células tumorales se sembraron en una placa de 24 pocillos a una densidad clonal de 300 células por ml de medio de cultivo. En cada pozo se observó colonias clonal ampliado. Esta serie de imágenes de campo claro demostrar cambios típicos durante un período de 10 días de la cultura. <img src="/files/ftp_upload/2086/2086fig5.jpg" alt="Figura 5" /> Figura 5. Análisis de la diferenciación de las células de meduloblastoma establecido. Cuando las células tumorales fueron cambiados de EGF / bFGF contiene medio libre de suero para DMEM/10% de SFB, YFP + células tumorales alterado de manera significativa su morphology y diferenciado en varios tipos celulares, incluyendo Tuj1 + o + NeuN las neuronas, las células GFAP + astroglial o CNPase + oligodendrocitos.

<ol>
	<li>Rossi, A., Caracciolo, V., Russo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma:+From+Molecular+Pathology+to+Therapy.">Medulloblastoma: From Molecular Pathology to Therapy.</a> Clin Cancer Res. 14 (4), 971-971 (2008).</li><li>Wechsler-Reya, R. J., Scott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Neuronal+Precursor+Proliferation+in+the+Cerebellum+by+Sonic+Hedgehog.">Control of Neuronal Precursor Proliferation in the Cerebellum by Sonic Hedgehog.</a> P. Neuron. 22 (1), 103-103 (1999).</li><li>Dahmane, N., Ruiz, A., Altaba, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+the+growth+and+patterning+of+the+cerebellum.">Sonic hedgehog regulates the growth and patterning of the cerebellum.</a> Development. 126 (14), 3089-3089 (1999).</li><li>Wallace, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purkinje-cell-derived+Sonic+hedgehog+regulates+granule+neuron+precursor+cell+proliferation+in+the+developing+mouse+cerebellum.">Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum.</a> Curr Biol. 9 (8), 445-445 (1999).</li><li>Fan, X., Eberhart, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medullablastoma+Stem+Cells.">Medullablastoma Stem Cells.</a> J Clin Oncol. 26 (17), 2821-2821 (2008).</li><li>Yoon, J. W., Gilbertson, R., Iannaccone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+Role+for+Hedgehog+Pathway+Activation+in+Desmoplastic+Medulloblastoma+by+Identifying+GLI1+Target+Genes.+International+journal+of+cancer.">Defining a Role for Hedgehog Pathway Activation in Desmoplastic Medulloblastoma by Identifying GLI1 Target Genes. International journal of cancer.</a> 124 (1), 109-109 (2009).</li><li>O'Dorisio, M. S., Khanna, G., Bushnell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+anatomic+and+molecularly+targeted+imaging+in+the+diagnosis+and+surveillance+of+embryonal+tumors+of+the+nervous+and+endocrine+systems+in+children.+Review.">Combining anatomic and molecularly targeted imaging in the diagnosis and surveillance of embryonal tumors of the nervous and endocrine systems in children. Review.</a> Cancer metastasis reviews. 27 (4), 665-665 (2008).</li><li>Corcoran, R. B., Bachar Raveh, T., Barakat, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+Growth+Factor+2+Is+Required+for+Progression+to+Advanced+Medulloblastoma+in+patched1+Heterozygous+Mice.">Insulin-like Growth Factor 2 Is Required for Progression to Advanced Medulloblastoma in patched1 Heterozygous Mice.</a> Cancer research. 68 (21), 8788-8788 (2008).</li><li>Gilbertson, R. J., Ellison, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Origins+of+Medulloblastoma+Subtypes.">The Origins of Medulloblastoma Subtypes.</a> Annual review of pathology. 3, 341-341 (2008).</li><li>Ward, R. J., Lee, L., Graham, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+CD15++Cancer+Stem+Cells+in+Patched-1+Deficient+Mouse+Medulloblastoma.">Multipotent CD15+ Cancer Stem Cells in Patched-1 Deficient Mouse Medulloblastoma.</a> Cancer research. 69 (11), 4682-4682 (2009).</li><li>Mao, J., Ligon, K. L., Rakhlin, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Somatic+Mouse+Model+to+Survey+Tumorigenic+Potential+Applied+to+the+Hedgehog+Pathway.">A Novel Somatic Mouse Model to Survey Tumorigenic Potential Applied to the Hedgehog Pathway.</a> Cancer research. 66, 10171-10171 (2006).</li><li>Han, Y. G., Kim, H. J., Dlugosz, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+and+opposing+roles+of+primary+cilia+in+medulloblastoma+development.">Dual and opposing roles of primary cilia in medulloblastoma development.</a> Nature medicine. 15 (9), 1062-1062 (2009).</li></ol>

Se describe una forma eficiente de aislar, enriquecer y mantener las células madre del tejido meduloblastoma tumor primario recuperado de ratones mutantes con constitutivamente activa la señalización de Hedgehog. Hemos encontrado que un paso crítico en el establecimiento exitoso de una madre sana tumor línea celular es el tratamiento Accutase utilizados durante la disociación del tejido del tumor primario. En nuestra experiencia, cuando el tejido del tumor primera disociación del cerebelo, 4 minutos de tratamiento Accutase 50% a 37 ° C, seguido de 3 minutos de pipeteado repetitivo con un Pipetman de 1 ml por lo general se traducirá en la siembra de primera con éxito. También es importante esperar hasta que se forman colonias considerable antes de volver a placa (Figura 1). Después de la primera siembra, el tratamiento Accutase ya no es necesario; pipeta repetitiva durante 3-4 minutos con una Pipetman de 1 ml es suficiente para desalojar y disociar grupos de células para la re-recubrimiento. No use más pequeños Pipetman volumen como las puntas finas se dañan las células ampliamente. Una línea celular establecida puede ser generada a partir de cada uno de los tejidos tumorales aisladas y que estas células son altamente proliferativas, con firmeza expresan varios marcadores de células madre neurales y pueden diferenciarse en dos tipos de células neuronales y gliales. Hemos llevado a cabo el trasplante ortotópico de estas células en ratones inmunodeficientes destinatario que posteriormente se meduloblastomas secundaria. Estas células tumorales en cultivo rara vez se exhiben diferenciación espontánea o apoptosis, características que son comunes a los métodos de cultivo neuroesfera. Este protocolo está diseñado para la propagación exitosa de las células madre tumorales derivadas de meduloblastomas primaria y por lo tanto, nos permitirá poner a prueba los efectos de los genes candidatos y los inhibidores químicos de erizo impulsado por el crecimiento de células tumorales y la conducta.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Neurobasal medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103049</td>
<td>&nbsp;</td>
<tr>
<td>hEGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0311</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0023</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>N2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502048</td>
<td>1X</td>
</tr>
<tr>
<td>B27(-RA)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12587010</td>
<td>1X</td>
</tr>
<tr>
<td>Accutase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1110501</td>
<td>50%</td>
</tr>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1393</td>
<td> 0.1%</td>
</tr>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>2mM</td>
</tr>
<tr>
<td>Primaria dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>087724C</td>
<td>&nbsp;</td>
<tr>
<td>Sox2 antibody</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MAB4343</td>
<td>Mouse, 1:1000</td>
</tr>
<tr>
<td>Nestin antibody</td>
<td>&nbsp;</td>
<td>DSHB</td>
<td>rat401</td>
<td>Mouse, 1:200</td>
</tr>
<tr>
<td>YFP antibody</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>A11122</td>
<td>Rabbit, 1:2000</td>
</tr>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Neuromics</td>
<td>CH22102</td>
<td>Chicken, 1:1000</td>
</tr>
<tr>
<td>Tuj1 antibody</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5076</td>
<td>Mouse, 1:2000</td>
</tr>
<tr>
<td>NeuN antibody</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MAB377</td>
<td>Mouse, 1:2000</td>
</tr>
<tr>
<td>CNPase antibody</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MAB326</td>
<td>Mouse, 1:1000</td>
</tr>		</tbody>
		</table>
		</div>

isolation, enrichment, and maintenance of medulloblastoma stem cells

Los tumores cerebrales se ha sugerido que posee una pequeña población de células madre que son la causa principal de la tumorogénesis. Neuroesfera ensayos han sido generalmente adoptado para estudiar la naturaleza de las células madre neuronales, incluyendo los derivados de los tejidos normales y tumorales. Sin embargo, cantidades apreciables de diferenciación y muerte celular son comunes en neuroesferas cultivadas probablemente debido a la sub-óptimas condiciones tales como la accesibilidad de todas las células dentro de los agregados ámbito de medio de cultivo. Meduloblastoma, el tumor infantil más común del SNC, se caracteriza por su rápida progresión y tendencia a expandirse por todo el cerebro-espinal eje con el resultado clínico deprimente. El meduloblastoma es un tumor neuroepitelial del cerebelo, que representan el 20% y 40% de los tumores de fosa intracraneal y posterior en la infancia, respectivamente 1. Es bien sabido ahora que la señalización de Shh estimula la proliferación de los precursores del cerebelo gránulo neurona (CGNPs) durante el desarrollo del cerebelo 2-4. Numerosos estudios utilizando modelos de ratón, en el que es constitutivamente la vía de Shh activa, han relacionado la señalización de Shh con meduloblastoma 5-9. Un informe reciente ha demostrado que un subgrupo de células derivadas de meduloblastoma Patched1 ratones LacZ / + son las células madre del cáncer, que son capaces de iniciar y propogating tumores 10. A continuación se describe un método eficiente para aislar, enriquecer y mantener las células madre tumorales derivadas de varios modelos de ratones de meduloblastoma, con la vía activada constitutivamente Shh debido a una mutación en el alisado (11, en ​​adelante nos referiremos como SmoM2), un GPCR que es fundamental para Shh vía de activación. En todos los tejidos meduloblastoma aislados, hemos sido capaces de establecer numerosas colonias que se reproducen más. Estas células expresan varios marcadores con firmeza las células madre neurales, como Nestin y Sox2, pueden someterse a los pases en serie (más de 20) y se clonación. Mientras que estas células madre tumorales cultivadas eran relativamente pequeñas, a menudo con bipoar nucleares de alta relación núcleo: citoplasma cuando se cultivan en condiciones que favorecen el crecimiento de células madre, que alteró dramáticamente su morfología, extendió múltiples procesos celulares, aplanado y se retiró del ciclo celular al cambiar a una celda medio de cultivo suplementado con suero fetal bovino al 10%. Más importante aún, estas células madre tumorales diferenciadas en Tuj1 + o + NeuN neuronas, GFAP + astrocitos y oligodendrocitos CNPase +, destacando así sus múltiples potencia. Además, estas células eran capaces de propagar meduloblastomas secundaria cuando ortotópicamente trasplantadas a ratones de acogida.

Watch this Scientific Journal Video about Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells at JoVE.com

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Este protocolo describe el aislamiento, el enriquecimiento y mantenimiento de las células madre tumorales meduloblastoma derivados de ratones mutantes con ectópico actividad de la vía sonic hedgehog.

Aislamiento, enriquecimiento y mantenimiento de las células madre meduloblastoma

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

isolation-enrichment-and-maintenance-of-medulloblastoma-stem-cells

Nanyang Technological University

Research

JoVE Journal

Neuroscience

16.2K vistas.  Vanderbilt University. Este protocolo describe el aislamiento, el enriquecimiento y mantenimiento de las células madre tumorales meduloblastoma derivados de ratones mutantes con ectópico actividad de la vía sonic hedgehog.

Video: Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma1-4. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker5, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered. Therefore, it is essential to determine whether there is an alternative medulloblastoma tumor cell-of-origin based on which cell-type specific therapeutic modality can be developed. To this end, intracranial orthotopic allografting of genetically marked tumor cell types followed by subsequent analyses of secondary tumor development in recipients will allow determination of the cellular origin of tumor-initiating cells. Here we describe the experimental protocol for intracranial orthotopic allografting of medulloblastoma cells derived from primary tumor tissue, and this procedure can also be used for transplanting cells from established cell lines.

This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.

Aloinjerto intracraneal ortotópico de células de meduloblastoma en ratones inmunodeficientes

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

Investigación

Neurociencias

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 ...

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 ...

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 ...

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 ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Aislamiento, cultivo y mantenimiento a largo plazo de Primaria mesencefálico neuronas dopaminérgicas a partir de cerebros de embriones de roedores

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

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.

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 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.

Aislamiento y cultivo de las células madre neurales embrionarias de ratón

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 ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.