Questo studio è stato supportato anche da finanziamenti della Vanderbilt-Ingram Cancer Center Support Grant (P30 CA068485), il Brain Tumor Foundation Infanzia e il National Institutes of Health (NS042205).

1. Micro-dissezione del tumore-cuscinetto Cervelletto, dissociazione del tessuto tumorale e placcatura <ol><li> Recupero del tessuto tumorale <ol><li> Sick topi portatori di medulloblastoma erano spesso runted, visualizzati idrocefalo e tipici sintomi neurologici, tra cui paralisi posteriore e l&#39;incapacità di riacquistare la postura in caso di ribaltamento. Per recuperare tessuto tumorale, eutanasia topi per inalazione di anidride carbonica. E &#39;importante non eseguire dislocazione cervicale, una procedura che genera una pressione al cranio posteriore e può compromettere l&#39;integrità del tessuto tumorale. </li><li> Decapitazione viene eseguita subito dopo la morte con un paio di forbici, la rimozione dei capelli e tessuto muscolare il più possibile per una buona visualizzazione del cranio. Pulire la superficie del cranio con Kimwipe imbevuto di etanolo al 95%. </li><li> Usare le forbici per tagliare bene un&#39;apertura lungo la linea mediana del cranio, e rimuovere il tessuto cranio con una pinzetta sottile, a questo punto l&#39;intero cervello compreso portatori di tumore cervelletto è esposto. </li><li> Mentre il cervello dei degli adulti sani visualizzare ben definite emisferi e del verme, il tumore del cervello dei topi portatori sono spesso allargata, amorfo con una superficie liscia e vasi sanguigni cospicua. Usando tecniche sterili, recuperare il tumore del cervelletto con delle pinzette e mettere in ghiaccio PBS freddo senza Mg2 + e Ca 2 +. </li></ol> Nota: tutti gli strumenti sono sterilizzati in etanolo al 95% prima dell&#39;uso. </li><li> Dissociazione del tessuto tumorale <ol><li> Trasferire il tessuto tumorale da PBS al 50% Accutase (diluito in PBS) che è circa 4 volte il volume del tessuto tumorale, tritare il tessuto con le forbici multa per 3 minuti a temperatura ambiente, seguita da incubazione a 37 ° C per 4 minuti , dopo di che il tessuto subisce pipeting ripetitivi con un 1 mL Pipetman per altri 3 minuti. Questo metodo dovrebbe produrre un insieme di celle singole e piccoli aggregati cellulari. </li><li> Diluire la sospensione cellulare di 3 volte con PBS e centrifugare per 5 minuti a 1000 g per agglomerare le cellule. Risospendere il pellet cellulare in acqua dolce neurali terreno di coltura delle cellule staminali e la piastra su una parabola di 60 gelatinizzato Primaria cultura millimetro di tessuto. Noi usiamo piatti Primaria per l&#39;attacco migliore in placcatura prima; passaggi successivi possono essere placcato su piatti regolare coltura dei tessuti. </li></ol></li></ol> Nota: piatti Cappotto cultura con 0,1% di gelatina per almeno 30 minuti. Preparare fresco medio delle cellule neurali staminali coltura composto medio Neurobasal con la glutamina, Pen-Strep, N2, B27, umano EGF (25 ng / ml) e di base FGF (25ng / mL). 2. Arricchimento, manutenzione ed espansione delle cellule tumorali da Medulloblastoma passaggi consecutivi Noi di solito ricevono molte colonie dopo 1 settimana di placcatura iniziale (Figura 1). Queste colonie può essere dissociata e saltuariamente su nuovi piatti gelatinizzato per l&#39;arricchimento della popolazione delle cellule tumorali. Primo cambio al terreno di coltura nuova, quindi è sufficiente utilizzare un 1-mL Pipetman per staccare meccanicamente colonie seguita da pipeting ripetitivi per 4 minuti fino ad ottenere una sospensione cellulare (non Accutase necessario). Controllare al microscopio per garantire che grumi grandi cellule sono state dissociate. Poi dispensare sospensione cellulare su nuovi piatti gelatinizzato per la coltura ulteriormente e passare attraverso la stessa procedura per i passaggi aggiuntivi. Cellule tumorali senza legami, cellule del sangue e altri tipi di cellule vengono rimosse da seriale ri-placcature, lasciando le cellule tumorali attaccato ad espandersi rapidamente. Cambiare mezzo di coltura il secondo giorno dopo la semina iniziale, e ogni due giorni dopo. 3. La colorazione di immunofluorescenza ed esame delle cellule in coltura 1. Crescere le cellule tumorali a lamelle di vetro Il giorno 1, mettere un coprioggetto di vetro in ciascuno di un 6-pozzetti, quindi aggiungere 0,1% di gelatina per 30 minuti a 37 ° C. Seme di circa 2-4X 10 5 cellule tumorali per mL. Le cellule devono allegare tutta la notte e le cellule staminali neurali medio cambiato il giorno 3. Colorazione viene eseguita il giorno 4. 2 colorazione immunofluorescenza Togliere terreno di coltura e le cellule lavare due volte con PBS freddo ghiaccio. Fissare le cellule con appena fatto il 4% PFA per 15 minuti. Brevemente lavare le cellule due volte con PBS e permeablize con 0,3% Triton (in PBS) per 5 minuti. Brevemente lavare le cellule due volte con PBS e blocco con siero di capra 10% per 40 minuti. Incubare le cellule con l&#39;anticorpo primario per 90 minuti, poi lavare le cellule tre volte con PBS, per 5 minuti ciascuno. Incubare le cellule con l&#39;anticorpo secondario per 60 minuti, poi lavare tre volte con PBS, 5 minuti ciascuno. Montaggio su vetrini coprioggetto con 5 ml di un mezzo acquoso di montaggio e di eseguire la microscopia confocale. 4. La differenziazione delle cellule tumorali Rimuovere nEURAL cellule staminali e medie brevemente lavare le cellule con PBS due volte. Aggiungere mezzo differenziazione al giorno 1, il cambiamento medio nel giorno 4 e determinare il livello di differenziazione da immunofluorescenza il giorno 7. Il mezzo di differenziazione è costituito da DMEM, Pen-Strep e il 10% di siero fetale bovino. 5. Rappresentante Risultati Morfologia risultati Dopo il trattamento Accutase e gentile pipeting ripetitivo, il tessuto tumorale dovrebbe essere per lo più dissociata in piccoli aggregati cellulari e cellule singole. Molte colonie di considerevoli dimensioni si possono osservare già 5 giorni dopo la placcatura iniziale, come mostrato dalla Figura 1. Cellule tumorali altamente proliferative sono spesso bi-polare, con elevato rapporto nucleare / citoplasma. Queste cellule si vedono di solito irradia da piccoli aggregati cellulari attaccati alla superficie gelatinizzata. Cellule del sangue, piccolo e rotondo, di solito sono rimosse dai successivi cambiamenti dei media e placcature di serie. Dopo vari passaggi, si può osservare uniformemente distribuiti, proliferativo Ki67 + cellule tumorali e la contaminazione poco con altri tipi di cellule (Figura 2). Espressione di marcatori di cellule staminali neurali, analisi clonale e differenziazione in linee multiple Per determinare se le cellule proliferative derivate da dissociato caratteristiche cerebellari mostrano tessuto tumorale di cellule staminali tumorali, abbiamo effettuato la caratterizzazione ulteriore espressione di marcatori di cellule staminali, l&#39;analisi clonale e la potenza differenziazione. Abbiamo scoperto che queste cellule tumorali isolate altamente esprimere marcatori di cellule staminali neurali come Sox2 e Nestin (Figura 3). Come abbiamo recuperate tessuti da SmoM2-YFP tumore, tutte le cellule tumorali esprimere YFP. In linea con un recente studio di reporting che ciglia primarie sono importanti per lo sviluppo di Shh percorso-dipendente medulloblastoma 12, SmoM2-YFP espressione è stata chiaramente localizzata alle ciglia di Sox2 + cellule tumorali (Figura 3). Quando placcato a densità clonale di 300 cellule per ml di terreno di coltura, abbiamo osservato l&#39;espansione clonale di una cellula tumorale solo ad una colonia di considerevoli dimensioni (Figura 4). Inoltre, queste cellule erano in grado di differenziarsi in vari tipi di cellule neuronali e gliali quando coltivate in pro-differenziazione condizioni (Figura 5). <img src="/files/ftp_upload/2086/2086fig1.jpg" alt="Figura 1" /> Figura 1. Morfologia di una colonia altamente proliferative dopo placcatura primo tessuto medulloblastoma dissociato. In genere, si forma una colonia consistente entro 5 giorni dalla semina iniziale di tessuto tumorale dissociato, e un rappresentante è illustrato qui in campo chiaro. Bi-polare, irradiare le cellule allungate da un denso aggregato di cellule fondamentali. Allegati alle cellule tumorali sono piccole, cellule rotonde rosse del sangue, che vengono gradualmente impoverito da placcature di serie. <img src="/files/ftp_upload/2086/2086fig2.jpg" alt="Figura 2" /> Figura 2. Morfologia delle cellule di medulloblastoma stabilita oltre tre passaggi. Questa immagine in campo chiaro mostra l&#39;aspetto tipico delle cellule medulloblastoma stabilito dopo diversi passaggi. Le cellule rimangono per lo più bi-polare, con alta attività / citoplasmatica rapporto. Mostriamo colorazione acetilati tubulina delle cellule tumorali, la maggior parte delle quali sono in bicicletta, come mostrato da una forte espressione di Ki67. <img src="/files/ftp_upload/2086/2086fig3.jpg" alt="Figura 3" /> Figura 3. Cellule di medulloblastoma stabilito esprimere marcatori di cellule staminali neurali. SmoM2-YFP segni di cellule che esprimono costitutivamente attiva Smo, quindi l&#39;attività pathway Shh. Tutte le cellule del medulloblastoma linee cellulari stabilito espresso YFP, e la maggior parte di loro co-espressi alti livelli di marcatori di cellule staminali neurali diverse, come ad esempio Nestin e Sox2. È interessante notare che quando abbiamo effettuato il rilevamento del segnale YFP con potenza laser minimo durante la microscopia confocale, abbiamo rilevato il segnale YFP concentrato nelle cellule cilia di Sox2 +. Questa osservazione è coerente con un 12 recente rapporto che ha descritto il ruolo essenziale delle ciglia per lo sviluppo di Shh percorso-dipendente medulloblastoma. <img src="/files/ftp_upload/2086/2086fig4.jpg" alt="Figura 4" /> Figura 4. Fondata cellule tumorali subiscono espansione clonale. Dopo la dissociazione in sospensione singola cellula, le cellule tumorali sono stati piastrati su un piatto ben 24 con una densità clonale di 300 cellule per ml di terreno di coltura. In ogni pozzetto abbiamo osservato le colonie clonale ampliato. Questa serie di immagini in campo chiaro dimostrare i cambiamenti tipici nel corso di un periodo di 10 giorni di cultura. <img src="/files/ftp_upload/2086/2086fig5.jpg" alt="Figura 5" /> Figura 5. Analisi di differenziazione delle cellule medulloblastoma stabilito. Quando le cellule tumorali sono passati da FEG / bFGF contenente mezzo privo di siero di FBS% DMEM/10, YFP + cellule tumorali significativamente alterato la loro morfologia e differenziato in vari tipi di cellule, incLuding Tuj1 NeuN + + o neuroni, le cellule GFAP + astrogliali o CNPase + oligodendrociti.

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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>

Nessun conflitto di interessi dichiarati.

Descriviamo un modo efficace per isolare, arricchire e mantenere le cellule staminali da medulloblastoma primario tessuto tumorale recuperato da topi mutanti con costitutivamente attiva di segnalazione Hedgehog. Abbiamo scoperto che un passo fondamentale in successo, che istituisce un sano linea di staminali delle cellule tumorali è il trattamento Accutase utilizzato durante la dissociazione del tessuto del tumore primario. Nella nostra esperienza, al momento della prima tessuto tumorale dissociare dal cervelletto, 4 m...

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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

Tumori del cervello sono state proposte di possedere una piccola popolazione di cellule staminali che sono la causa principale di tumorigenesi. Saggi neurosfere sono state generalmente adottate per studiare la natura delle cellule staminali neurali, compresi quelli derivati ​​da tessuti normali e tumorali. Tuttavia, quantità apprezzabili di differenziazione e morte cellulare sono comuni nelle neurosfere colto probabilmente dovuto alla non ottimale condizione come l&#39;accessibilità di tutte le cellule all&#39;interno degli aggregati sfera di terreno di coltura. Medulloblastoma, il più comune tumore pediatrico sistema nervoso centrale, è caratterizzata da rapida progressione e la tendenza a diffondersi lungo l&#39;intero cervello-spinale, con triste risultato clinico. Medulloblastoma è un tumore neuroepiteliale del cervelletto, che rappresentano il 20% e il 40% dei tumori intracranici e fossa posteriore durante l&#39;infanzia, rispettivamente 1. E &#39;ormai noto che Shh segnalazione stimola la proliferazione dei precursori neuronali dei granuli cerebellari (CGNPs) durante lo sviluppo del cervelletto 2-4. Numerosi studi utilizzando modelli del mouse, in cui il percorso Shh è costitutivamente attivata, hanno collegato segnalazione Shh con medulloblastoma 5-9. Un recente rapporto ha mostrato che un sottogruppo di cellule derivate da medulloblastoma Patched1 topi LacZ / + sono le cellule staminali tumorali, che sono in grado di avviare e propogating tumori 10. Qui si descrive un metodo efficace per isolare, arricchire e mantenere le cellule staminali tumorali derivanti da alcuni modelli murini di medulloblastoma, con costitutivamente attivato percorso Shh a causa di una mutazione in Smoothened (11, di cui esso contenute come SmoM2), un GPCR, che è fondamentale per Shh percorso di attivazione. In ogni tessuto medulloblastoma isolato, siamo stati in grado di stabilire numerose colonie altamente proliferative. Queste cellule robustamente espresso diversi marcatori di cellule staminali neurali come Nestin e Sox2, possono subire passaggi consecutivi (maggiore di 20) e sono stati clonogenica. Mentre queste colture di cellule staminali tumorali sono relativamente piccole, spesso bipoar nucleare con elevato rapporto citoplasmatico quando coltivate in condizioni favoriscono la crescita delle cellule staminali, hanno drasticamente modificato la loro morfologia, esteso più processi cellulari, appiattite e si ritirò dal ciclo cellulare dopo il passaggio a una cella mezzo di coltura supplementato con 10% di siero fetale bovino. Ancora più importante, queste cellule staminali tumorali differenziate in Tuj1 NeuN + + o neuroni, astrociti GFAP + e + CNPase oligodendrociti, evidenziando la loro multi-potenza. Inoltre, queste cellule erano in grado di propagare medulloblastomi secondario quando ortotopicamente trapiantate in topi di accoglienza.

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

Questo protocollo descrive l&#39;isolamento, l&#39;arricchimento e il mantenimento delle cellule tumorali medulloblastoma staminali derivate da topi mutanti con percorso di attività ectopica di Sonic hedgehog.

Isolamento, di arricchimento e manutenzione delle cellule staminali Medulloblastoma

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

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16.2K Views.  Vanderbilt University. Questo protocollo descrive l'isolamento, l'arricchimento e il mantenimento delle cellule tumorali medulloblastoma staminali derivate da topi mutanti con percorso di attività ectopica di 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.

Intracranica allotrapianto ortotopico di cellule di medulloblastoma in topi immunocompromessi

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

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Neuroscienze

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 ed espansione della cellule staminali neurali del mouse utilizzando il saggio neurosfere

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.

L&#39;isolamento e la coltura di cellule staminali neurali Crest di follicoli dei capelli umani

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.

Il saggio neuroblasti: un test per la generazione e arricchimento delle cellule progenitrici neuronali di differenziare i Progeny cellule staminali neurali in citometria a flusso

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 la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

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 lungo termine Manutenzione di primaria mesencefalico neuroni dopaminergici Da Brains embrionali dei roditori

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 la coltura di adulti neurali cellule staminali dal Subcallosal Zona mouse

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 coltura di cellule staminali neurali embrionali del Mouse

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.

Un modello scalabile per studiare gli effetti della lesione da forza smussata nel pesce zebra 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 come strumento di apprendimento associativo per la valutazione cognitiva negli studi sull'apprendimento e sulla memoria utilizzando Zebrafish adulto

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