Este estudo foi suportado por concessões do Vanderbilt-Ingram Cancer Center de Suporte Grant (P30 CA068485), a Childhood Foundation tumor cerebral e os Institutos Nacionais de Saúde (NS042205).

1. Micro dissecção de Tumor-rolamento Cerebelo, dissociação do tecido tumoral e Galvanização <ol><li> Recuperação do tecido tumoral <ol><li> Meduloblastoma tendo doente camundongos foram muitas vezes runted, exibido hidrocefalia e típicos sintomas neurológicos, incluindo paralisia posterior e insuficiência de recuperar a postura, quando capotou. Para recuperar tecido tumoral, euthanize ratos por inalação de dióxido de carbono. É importante não realizar deslocamento cervical, um procedimento que gera pressão para a posterior do crânio e pode comprometer a integridade do tecido do tumor. </li><li> Decapitação é realizada imediatamente após a morte usando um par de tesouras, a remoção de pêlos e tecido muscular, tanto quanto possível para uma boa visualização do crânio. Limpe a superfície do crânio com Kimwipe embebido com álcool 95%. </li><li> Use uma tesoura fina para cortar uma abertura ao longo da linha média do crânio, e remover o tecido usando uma pinça fina crânio, altura em que todo o cérebro, incluindo tumor-rolamento cerebelo é exposto. </li><li> Enquanto o cerebelo de adultos saudáveis ​​mostrar bem definidas hemisférios e vermis, o cerebelo de camundongos portadores de tumor são muitas vezes alargada, amorfa com uma superfície lisa e vasos sanguíneos visíveis. Usando técnicas estéreis, recuperar o tumor cerebelar usando uma pinça e coloque em PBS gelado sem Mg2 + e Ca 2 +. </li></ol> Nota: todos os instrumentos são esterilizados em etanol 95% antes de usar. </li><li> Dissociação do tecido tumoral <ol><li> Transferir o tecido tumoral a partir de PBS para Accutase 50% (diluído em PBS), que é cerca de 4 vezes o volume do tecido tumoral, mince o tecido com uma tesoura bem por 3 minutos em temperatura ambiente, seguido de incubação a 37 ° C por 4 minutos , após o qual o tecido passa por pipetagem repetitiva com um Pipetman de 1 ml para mais 3 minutos. Este método deve render uma mistura de células isoladas e pequenos agregados celulares. </li><li> Diluir a suspensão celular 3 vezes com PBS e centrifugar por 5 minutos a 1000 g para agregar as células. Ressuspender o pellet celular em meio de cultura fresco neural de células-tronco e em uma placa de 60 milímetros gelatinizado Primaria prato de cultura de tecidos. Usamos pratos Primaria para fixação reforçada em chapeamento em primeiro lugar; passagens subseqüentes podem ser semeados em placas de cultura de tecido normal. </li></ol></li></ol> Nota: pratos Brasão cultura com 0,1% de gelatina por pelo menos 30 minutos. Prepare-tronco neurais fresco meio de cultura celular consistindo de médio Neurobasal com glutamina, Pen-Strep, N2, B27, EGF humano (25 ng / mL) e FGF básico (25 ng / mL). 2. Manutenção, enriquecimento e expansão das células tumorais Meduloblastoma por passagens seriadas Costumamos ter muitas colônias após 1 semana de o início do cultivo (Figura 1). Estas colônias podem ser dissociadas e replated em novos pratos gelatinizado para o enriquecimento da população de células tumorais. Primeira mudança de meio de cultura novo, então simplesmente use um Pipetman de 1 ml para separar mecanicamente colônias seguido por pipetagem repetitiva por 4 minutos para produzir uma suspensão celular (não Accutase necessário). Verificar sob um microscópio para assegurar que aglomerados de grandes células foram dissociadas. Então dispensar suspensão de células em novos pratos gelatinizado para cultura ainda mais e atravessar o mesmo procedimento para passagens adicionais. Células tumorais desapegado, células sanguíneas e outros tipos de células são removidas por série re-chapeamentos, deixando as células do tumor ligado a expandir-se rapidamente. Alterar meio de cultura, no segundo dia após a semeadura inicial, e todos os outros dias depois. 3. A coloração de imunofluorescência e Exame de células cultivadas 1. Cultivar células tumorais em lamínulas No dia 1, coloque uma lamela de vidro em cada um uma placa 6-bem, em seguida, adicionar 0,1% de gelatina por 30 minutos a 37 ° C. Semente de cerca de 2-4X 10 5 células tumorais por ml. As células devem anexar noite eo meio células-tronco neurais mudou no dia 3. Coloração é realizada no dia 4. 2 coloração imunofluorescência Remova o meio de cultura e lave as células duas vezes com PBS gelado. Fixar as células com recém-made PFA 4% por 15 minutos. Brevemente lavar as células duas vezes com PBS e permeablize com Triton 0,3% (em PBS) por 5 minutos. Brevemente lavar as células duas vezes com PBS e bloquear com soro de cabra 10% por 40 minutos. Incubar as células com o anticorpo primário por 90 minutos, depois lave as células três vezes com PBS, por 5 minutos cada. Incubar as células com anticorpo secundário por 60 minutos, depois lavar três vezes com PBS, 5 minutos cada. Monte lamínulas em lâminas com 5 mL de um meio aquoso de montagem e realização de microscopia confocal. 4. Diferenciação das células tumorais Remover nEURAL meio de células-tronco e brevemente lavar as células com PBS duas vezes. Adicionar médio diferenciação no dia 1, a mudança média no dia 4 e determinar o nível de diferenciação por imunofluorescência no dia 7. O meio de diferenciação consiste em DMEM, Pen-Strep e 10% de soro fetal bovino. 5. Resultados representante Resultados morfologia Após o tratamento Accutase pipetagem repetitiva e suave, o tecido do tumor deve ser dissociadas em sua maioria em pequenos agregados celulares e células individuais. Muitas colônias considerável pode ser observado logo em 5 dias após o início do cultivo, como mostrado na Figura 1. Células tumorais altamente proliferativa são muitas vezes bi-polar com relação nuclear / cytoplasmic elevada. Estas células são vistos tipicamente irradiam de pequenos agregados celulares ligados à superfície gelatinizada. Células do sangue, pequenas e redondas, são normalmente removidas por alterações posteriores mídia e chapeamentos serial. Depois de várias passagens, pode-se observar uniformemente distribuída, proliferativa Ki67 + células tumorais e contaminação pouco com outros tipos de células (Figura 2). Expressão de marcadores de células-tronco neurais, análise clonal e diferenciação em múltiplas linhagens Para determinar se as células proliferativas derivado de tumor cerebelar dissociada características do tecido show de células-tronco tumorais, foi realizada a caracterização adicional pela expressão marcador de células-tronco, a análise clonal e potência de diferenciação. Descobrimos que essas células tumorais isoladas altamente expressam marcadores de células-tronco neurais, tais como Sox2 e nestina (Figura 3). À medida que recuperados a partir de tecidos SmoM2-YFP tumor, todas as células do tumor expressar YFP. De acordo com um recente estudo relatando que cílios são importantes para o desenvolvimento de meduloblastoma caminho-dependente Shh 12, SmoM2-YFP expressão foi claramente localizada aos cílios de Sox2 + células tumorais (Figura 3). Quando plaqueadas na densidade clonal de 300 células por mL de meio de cultura, observou-se expansão clonal de uma célula de tumor único para uma colônia de tamanho considerável (Figura 4). Além disso, essas células foram capazes de se diferenciar em vários tipos de células neuronais e gliais, quando cultivado nas condições pro-diferenciação (Figura 5). <img src="/files/ftp_upload/2086/2086fig1.jpg" alt="Figura 1" /> Figura 1. Morfologia de uma colônia altamente proliferativa após o plaqueamento primeiro de tecido meduloblastoma dissociados. Geralmente, uma forma considerável colônia dentro de 5 dias após a semeadura inicial de tecido tumoral dissociados, e um representante é ilustrada aqui em campo claro. Bi-polar, irradiar células alongadas a partir de um agregado denso núcleo da célula. Anexado às células tumorais são pequenas, redondas glóbulos vermelhos, que são gradualmente esgotado por chapeamentos serial. <img src="/files/ftp_upload/2086/2086fig2.jpg" alt="Figura 2" /> Figura 2. Morfologia das células meduloblastoma estabelecido além de três passagens. Esta imagem de campo claro mostra a aparência típica de células meduloblastoma estabelecido após várias passagens. As células permanecem em sua maioria bi-polar com relação nuclear / cytoplasmic elevada. Mostramos coloração tubulina acetilada das células tumorais, a maioria dos quais de bicicleta, como mostrado pela expressão Ki67 forte. <img src="/files/ftp_upload/2086/2086fig3.jpg" alt="Figura 3" /> Figura 3. Meduloblastoma células expressam marcadores estabelecidos células-tronco neurais. SmoM2-YFP marcas células que expressam Smo constitutivamente ativa, portanto, atividade da via Shh. Todas as células das linhas celulares estabelecidas meduloblastoma expressa YFP, ea maioria deles co-expressa altos níveis de vários marcadores de células-tronco neurais, tais como nestina e Sox2. Curiosamente, quando realizamos YFP detecção de sinal com potência do laser mínima durante microscopia confocal, detectamos sinal YFP concentrados nos cílios das células Sox2 +. Esta observação é consistente com um relatório recente que 12 descreveu o papel essencial dos cílios no desenvolvimento de meduloblastoma via dependente de Shh. <img src="/files/ftp_upload/2086/2086fig4.jpg" alt="Figura 4" /> Figura 4. Fundada células tumorais sofrer expansão clonal. Depois de dissociação em suspensão única célula, células tumorais foram semeadas em uma placa de 24 poços a uma densidade clonal de 300 células por ml de meio de cultura. Em cada poço observamos colônias clonalmente expandida. Esta série de imagens de campo claro demonstrar alterações típicas ao longo de um período de 10 dias da cultura. <img src="/files/ftp_upload/2086/2086fig5.jpg" alt="Figura 5" /> Figura 5. Analisa a diferenciação de células meduloblastoma estabelecida. Quando as células tumorais foram trocados a partir de EGF / bFGF contendo soro livre de médio a DMEM/10% FBS, YFP + células tumorais alteraram significativamente a sua morfologia e diferenciada em vários tipos celulares, incLuding Tuj1 + ou + NeuN neurônios, células GFAP + + CNPase astroglial ou oligodendrócitos.

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	<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>

Não há conflitos de interesse declarados.

Descrevemos uma forma eficiente para isolar, enriquecer e manter as células-tronco do tecido meduloblastoma tumor primário recuperados de camundongos mutantes com constitutivamente ativa Hedgehog sinalização. Descobrimos que um passo crítico no sucesso estabelecer uma linha saudável tumor de células-tronco é o tratamento Accutase usado durante a dissociação do tecido do tumor primário. Em nossa experiência, quando o tecido do tumor primeiro dissociar do cerebelo, 4 minutos de tratamento Accutase 50% a 37 ° ...

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		<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

Tumores cerebrais têm sido sugeridos para possuir uma pequena população de células-tronco que são a causa raiz de tumorigênese. Neurosphere ensaios têm sido geralmente adotado para estudar a natureza das células-tronco neurais, incluindo aquelas derivadas de tecidos normais e tumorais. No entanto, quantidades apreciáveis ​​de diferenciação e morte celular são comuns na cultura neurospheres provavelmente devido ao sub-ótimas condições tais como a acessibilidade de todas as células dentro de agregados esfera para meio de cultura. Meduloblastoma, o tumor mais comum do SNC pediátrica, é caracterizada pela sua rápida progressão e tendência a se espalhar ao longo do eixo cérebro-espinhal inteira com o resultado clínico sombrio. Meduloblastoma é um tumor neuroepiteliais do cerebelo, responsável por 20% e 40% dos tumores intracranianos e fossa posterior na infância, respectivamente 1. É agora bem estabelecido que a sinalização Shh estimula a proliferação de precursores granulares do cerebelo neurônio (CGNPs) durante o desenvolvimento do cerebelo 2-4. Numerosos estudos utilizando modelos de ratos, em que a via de Shh é constitutivamente ativado, ligaram sinalização Shh com meduloblastoma 5-9. Um relatório recente mostrou que um subconjunto de células derivadas de meduloblastoma Patched1 ratos LacZ / + são as células-tronco cancerosas, que são capazes de iniciar e propogating tumores 10. Aqui nós descrevemos um método eficiente para isolar, enriquecer e manter células-tronco tumorais derivados de modelos do rato várias meduloblastoma, com constitutivamente ativado via de Shh devido a uma mutação no Smoothened (11, doravante referida como SmoM2), um GPCR, que é fundamental para Shh ativação da via. Em todos os tecidos meduloblastoma isoladas, foram capazes de estabelecer numerosas colônias altamente proliferativa. Estas células robustamente expressa vários marcadores de células-tronco neurais, tais como nestina e Sox2, pode sofrer passagens em série (maior que 20) e foram clonogênica. Embora essas células-tronco cultivadas tumor eram relativamente pequenos, muitas vezes com bipoar nuclear elevada relação entre citoplasmática quando cultivadas em condições favorecendo o crescimento de células-tronco, que alterou drasticamente sua morfologia, estendida vários processos celulares, achatada e retirou-se do ciclo celular após a mudança para uma célula meio de cultura suplementado com 10% de soro fetal bovino. Mais importante, estas células-tronco tumorais diferenciado em Tuj1 + ou + NeuN neurônios, astrócitos GFAP + e + CNPase oligodendrócitos, ressaltando assim a sua potência multi-. Além disso, essas células eram capazes de propagar meduloblastomas secundário quando ortotopicamente transplantadas em camundongos host.

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 descreve o isolamento, enriquecimento e manutenção das células do tumor meduloblastoma-tronco derivadas de camundongos mutantes com ectópica atividade da via sonic hedgehog.

Enriquecimento de isolamento, e Manutenção de Células-Tronco 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

Research

JoVE Journal

Neuroscience

16.2K visualizações.  Vanderbilt University. Este protocolo descreve o isolamento, enriquecimento e manutenção das células do tumor meduloblastoma-tronco derivadas de camundongos mutantes com ectópica atividade da via sonic hedgehog.

Vídeo: 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.

Allografting ortotópico intracraniana de Células Meduloblastoma em camundongos imunocomprometidos

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

Neurociência

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.

Isolamento e Expansão do Rato Adulto células-tronco neurais utilizando o ensaio Neurosphere

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 ...

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.

Isolamento e Cultura de células da crista neural-tronco de folículos capilares humanos

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.

O Ensaio neuroblasto: um ensaio para a Geração e Enriquecimento de células progenitoras neuronais de Diferenciando Progeny Neural Stem Cell utilizando citometria de fluxo

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.

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

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.

Isolamento, cultura e Long-Term Manutenção da Primária mesencefálico Dopaminergic neurônios dos cérebros de roedores Embryonic

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.

Isolamento e Cultura de células estaminais adultas neurais do Subcallosal Zona Rato

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.

Isolamento e cultura de células-tronco neurais embrionárias de rato

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.

Um modelo escalável para estudar os efeitos da lesão por força bruta em zebrafish adulto

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 ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Shuttle Box Assay como uma ferramenta de aprendizagem associativa para avaliação cognitiva em estudos de aprendizagem e memória usando zebrafish adulto

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...