Cette étude a été soutenue par des subventions du Vanderbilt-Ingram Cancer Center Grant Support (P30 CA068485), la Fondation des tumeurs cérébrales enfance et les National Institutes of Health (NS042205).

1. Micro-dissection de la tumeur portant Cervelet, dissociation du tissu tumoral et placage <ol><li> Récupération de tissu tumoral <ol><li> Malade médulloblastome chez des souris porteuses sont souvent chétifs, affiché hydrocéphalie et typique des symptômes neurologiques, y compris paralysie des membres postérieurs et l&#39;incapacité à retrouver une posture en cas de retournement. Pour récupérer le tissu tumoral, euthanasier les souris par inhalation de dioxyde de carbone. Il est important de ne pas effectuer dislocation cervicale, une procédure qui génère une pression sur le crâne postérieur et peut compromettre l&#39;intégrité des tissus tumoraux. </li><li> Décapitation est effectuée immédiatement après la mort en utilisant une paire de ciseaux, enlever les poils et les tissus musculaires, autant que possible pour une bonne visualisation du crâne. Nettoyer la surface du crâne avec Kimwipe imbibé d&#39;éthanol à 95%. </li><li> Utilisez des ciseaux pour couper fines une ouverture le long de la ligne médiane du crâne, et éliminer les tissus du crâne avec une pince fine, à quel point l&#39;ensemble du cerveau, y compris porteurs d&#39;une tumeur du cervelet est exposée. </li><li> Alors que les cervelets des adultes sains d&#39;affichage bien définis hémisphères et du vermis, les cervelets de souris porteuses de tumeurs sont souvent élargie, amorphe avec une surface lisse et les vaisseaux sanguins visibles. En utilisant des techniques stériles, extraire la tumeur du cervelet en utilisant une pince à épiler et le lieu de PBS glacé, sans Mg 2 + et Ca 2 +. </li></ol></li> Remarque: tous les instruments sont stérilisés dans de l&#39;éthanol à 95% avant utilisation. <li> La dissociation du tissu tumoral <ol><li> Transférer le tissu tumoral du PBS pour Accutase 50% (dilué dans du PBS), qui est environ 4 fois le volume du tissu tumoral, émincer les tissus avec des ciseaux fins pour 3 minutes à température ambiante, suivie d&#39;une incubation à 37 ° C pendant 4 minutes , après quoi le tissu subit pipetage répétitifs avec un Pipetman de 1 ml pour 3 minutes supplémentaires. Cette méthode devrait donner un mélange de cellules individuelles et de petits agrégats cellulaires. </li><li> Diluer la suspension cellulaire 3 fois avec du PBS et centrifuger pendant 5 minutes à 1000 g pour sédimenter les cellules. Resuspendre la cellule dans l&#39;eau douce moyennes tiges de culture de cellules neurales et plaque granulés sur une parabole de 60 mm gélatinisé Primaria culture de tissus. Nous utilisons des plats Primaria pour la fixation améliorée au plaquage première passages ultérieurs peuvent être étalés sur des boîtes de culture tissulaire régulière. </li></ol></li></ol> Remarque: des boîtes de culture Manteau avec 0,1% de gélatine pendant au moins 30 minutes. Préparer une solution fraîche moyennes neuronaux de culture de cellules souches composé de milieu Neurobasal avec de la glutamine, Pen-Strep, N2, B27, EGF humain (25 ng / ml) et FGF basique (25 ng / ml). 2. Enrichissement, entretien et l&#39;expansion des cellules tumorales par des passages en série Médulloblastome Nous obtenons habituellement de nombreuses colonies après 1 semaine de plaquage initial (figure 1). Ces colonies peuvent être dissociés et sur de nouveaux plats réétalées gélatinisé pour l&#39;enrichissement de la population de cellules tumorales. Premier changement au nouveau milieu de culture, il suffit d&#39;utiliser un Pipetman 1 mL mécaniquement détacher colonies suivie par pipetage répétitifs pendant 4 minutes pour obtenir une suspension cellulaire (pas Accutase nécessaire). Vérifiez sous un microscope pour s&#39;assurer que les mottes à grandes cellules ont été dissociées. Puis passer la suspension cellulaire sur de nouveaux plats gélatinisé pour cultiver plus loin et passer par la même procédure pour les passages supplémentaires. Seules les cellules tumorales, les cellules sanguines et d&#39;autres types cellulaires sont éliminés par série re-placages, laissant les cellules tumorales attachés à développer rapidement. Changer milieu de culture sur le deuxième jour suivant l&#39;ensemencement initial, et chaque jour d&#39;autres par la suite. 3. La coloration par immunofluorescence et examen des cellules cultivées 1. Cultiver des cellules tumorales sur des lamelles de verre Au jour 1, lamelle de verre placer un dans chacun d&#39;une plaque à 6 puits, puis ajouter 0,1% de gélatine pendant 30 minutes à 37 ° C. Graines d&#39;environ 2-4x 10 5 cellules tumorales par mL. Les cellules devraient attacher la nuit et le milieu des cellules souches neurales changé le jour 3. La coloration est réalisée sur 4 jours. 2 La coloration par immunofluorescence Retirer milieu de culture et les cellules laver deux fois avec PBS glacé. Fixer les cellules avec des fraîchement faite PFA 4% pendant 15 minutes. Brièvement laver les cellules deux fois avec du PBS et permeablize avec Triton 0,3% (dans du PBS) pendant 5 minutes. Brièvement laver les cellules deux fois avec du PBS et de bloquer avec du sérum de chèvre 10% pendant 40 minutes. Incuber les cellules avec l&#39;anticorps primaire pendant 90 minutes, puis lavez les cellules trois fois avec PBS, pendant 5 minutes chacune. Incuber les cellules avec un anticorps secondaire pendant 60 minutes, puis laver trois fois avec du PBS, 5 minutes chacun. Mont lamelles sur des lames avec 5 ml d&#39;un milieu aqueux de montage et d&#39;effectuer la microscopie confocale. 4. La différenciation des cellules tumorales Retirer neurones moyennes de cellules souches et brièvement laver les cellules avec du PBS deux fois. Ajouter milieu de différenciation sur le jour 1, le changement moyen au jour 4 et déterminer le niveau de différenciation par immunofluorescence au jour 7. Le milieu de différenciation consiste DMEM, Pen-Strep et 10% de sérum fœtal bovin. 5. Les résultats représentatifs Morphologie des résultats Après le traitement Accutase et doux pipetage répétitifs, le tissu tumoral devrait être principalement dissocié en petits agrégats cellulaires et des cellules individuelles. Beaucoup de colonies considérables peuvent être observés dès 5 jours après l&#39;étalement initial, comme le montre la figure 1. Cellules tumorales fortement prolifératives sont souvent bi-polaire avec un ratio nucléaire / cytoplasmique élevé. Ces cellules sont généralement vus rayonnant à partir de petits agrégats cellulaires attachés à la surface gélatinisé. Les cellules sanguines, petites et rondes, sont généralement enlevés par les changements de supports suivants et les placages de série. Après plusieurs passages, on peut observer uniformément répartie, prolifératif Ki67 + cellules tumorales et la contamination peu avec d&#39;autres types cellulaires (figure 2). Expression des marqueurs des cellules souches neurales, analyse clonale et différenciation en multiples lignées Afin de déterminer si les cellules proliférantes dérivées de dissocier caractéristiques de la tumeur du cervelet montrent tissu de cellules souches tumorales, nous avons effectué une caractérisation plus poussée par l&#39;expression du marqueur de cellules souches, l&#39;analyse clonale et la puissance de différenciation. Nous avons constaté que ces cellules tumorales isolées très explicite marqueurs des cellules souches neurales telles que Sox2 et nestine (figure 3). Comme nous l&#39;avons récupéré à partir des tissus tumoraux SmoM2-YFP, toutes les cellules tumorales expriment YFP. Conformément à une récente étude indiquant que les cils primaires sont importants pour le développement de la voie Shh-dépendant médulloblastome 12, SmoM2-YFP d&#39;expression a été clairement localisé les cils des cellules tumorales Sox2 + (figure 3). Lorsque la densité plaquées à clonale de 300 cellules par mL de milieu de culture, nous avons observé l&#39;expansion clonale d&#39;une cellule tumorale unique d&#39;une colonie importante (figure 4). En outre, ces cellules ont été capables de se différencier en divers types de cellules neuronales et gliales lorsqu&#39;elles sont cultivées dans des conditions pro-différenciation (figure 5). <img src="/files/ftp_upload/2086/2086fig1.jpg" alt="Figure 1" /> Figure 1. Morphologie d&#39;une colonie hautement proliférative après le placage d&#39;abord des tissus médulloblastome dissociées. Généralement, une colonie importante des formes dans les 5 jours après l&#39;ensemencement initial de tissu tumoral dissocié, et un représentant est illustré ici dans le champ lumineux. Bi-polaire, rayonnent des cellules allongées d&#39;un agrégat de cellules noyau dense. Attaché aux cellules tumorales sont petites, rondes globules rouges, qui sont progressivement épuisés par placages de série. <img src="/files/ftp_upload/2086/2086fig2.jpg" alt="Figure 2" /> Figure 2. Morphologie des cellules de médulloblastome établis au delà de trois passages. Cette image en champ clair montre l&#39;aspect typique des cellules de médulloblastome établie après plusieurs passages. Les cellules restent essentiellement bi-polaire de haute nucléaire / cytoplasmique ratio. Nous montrons coloration tubuline acétylée de cellules tumorales, dont la plupart sont le vélo, comme indiqué par une forte Ki67 expression. <img src="/files/ftp_upload/2086/2086fig3.jpg" alt="Figure 3" /> Figure 3. Cellules de médulloblastome Fondée expresse marqueurs des cellules souches neurales. SmoM2-YFP marques cellules exprimant constitutivement active Smo, donc activité de la voie Shh. Toutes les cellules des lignées cellulaires établies médulloblastome exprimé YFP, et la plupart d&#39;entre eux co-exprimé des niveaux élevés de différents marqueurs des cellules souches neurales, tels que la nestine et Sox2. Fait intéressant, lorsque nous avons effectué la détection du signal YFP avec la puissance laser minimale pendant la microscopie confocale, nous avons détecté le signal YFP concentrés dans les cils des cellules + Sox2. Cette observation est cohérente avec un rapport récent que 12 décrit le rôle essentiel des cils dans le développement de la voie Shh-dépendant médulloblastome. <img src="/files/ftp_upload/2086/2086fig4.jpg" alt="Figure 4" /> Figure 4. Fondée cellules tumorales subissent une expansion clonale. Après dissociation en suspension cellulaire unique, les cellules tumorales ont été étalées sur une plaque de 24 puits-à une densité clonale de 300 cellules par ml de milieu de culture. Dans chaque puits, nous avons observé des colonies par clonage élargi. Cette série d&#39;images en champ clair démontrer les changements typiques sur une période de 10 jours de culture. <img src="/files/ftp_upload/2086/2086fig5.jpg" alt="Figure 5" /> Figure 5. Analyses de différenciation des cellules de médulloblastome établie. Lorsque les cellules tumorales sont passés du FEM / bFGF contenant un milieu sans sérum à DMEM/10% de FBS, YFP + cellules tumorales sensiblement modifié leur morphology et différencié dans différents types cellulaires, y compris Tuj1 + ou + NeuN neurones, les cellules GFAP + astrogliales ou CNPase + oligodendrocytes.

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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>

Nous décrivons une façon efficace d&#39;isoler, d&#39;enrichir et de maintenir les cellules souches du tissu médulloblastome tumeur primaire récupérées à partir des souris mutantes avec constitutivement active de signalisation Hedgehog. Nous avons constaté qu&#39;un étape critique dans la réussite d&#39;un lignée tumorale des cellules souches saines est le traitement utilisé pendant Accutase la dissociation du tissu tumoral primaire. Dans notre expérience, lors de la première tissu tumoral dissociant du ce...

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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

Les tumeurs cérébrales ont été proposés à posséder une petite population de cellules souches qui sont à l&#39;origine de la tumorigenèse. Dosages Neurosphère ont été généralement adoptée pour étudier la nature des cellules souches neurales, y compris ceux issus de tissus normaux et tumoraux. Cependant, des quantités appréciables de différenciation et mort cellulaire sont communs dans neurosphères culture probablement en raison de sous-optimale état tels que l&#39;accessibilité de toutes les cellules dans les agrégats sphère au milieu de culture. Médulloblastome, la plus fréquente des tumeurs pédiatriques du SNC, est caractérisé par sa progression rapide et tendance à se propager le long de la totalité du cerveau-rachidien axe avec le résultat clinique lamentable. Le médulloblastome est une tumeur neuro-du cervelet, comptant pour 20% et 40% des tumeurs intracrâniennes et la fosse postérieure dans l&#39;enfance, respectivement 1. Il est maintenant bien établi que la signalisation de Shh stimule la prolifération des précurseurs de neurones du cervelet granules (CGNPs) pendant le développement du cervelet 2-4. De nombreuses études utilisant des modèles de souris, dans lequel la voie Shh est constitutivement activé, ont lié la signalisation de Shh avec médulloblastome 5-9. Un récent rapport a montré que sous-ensemble de cellules provenant de souris médulloblastome LacZ Patched1 / + sont des cellules souches cancéreuses, qui sont capables d&#39;initier et propogating tumeurs 10. Nous décrivons ici une méthode efficace pour isoler, d&#39;enrichir et de maintenir les cellules souches tumorales dérivées de plusieurs modèles murins de médulloblastome, avec voie Shh constitutivement activé en raison d&#39;une mutation dans Smoothened (11, Héraion mentionné que SmoM2), un RCPG qui est essentiel pour Shh activation de la voie. Dans tous les tissus médulloblastome isolés, nous avons été en mesure d&#39;établir de nombreuses colonies très prolifératives. Ces cellules robuste exprimé plusieurs marqueurs des cellules souches neurales telles que Nestin et Sox2, peuvent subir des passages en série (plus de 20) et ont été clonogénique. Bien que ces cellules souches tumorales en culture ont été relativement faibles, souvent avec des armes nucléaires bipoar élevé par rapport au cytoplasme lorsqu&#39;elles sont cultivées dans des conditions favorisant la croissance des cellules souches, ils radicalement modifié leur morphologie, prolongée de plusieurs processus cellulaires, aplatie et se retire du cycle cellulaire lors de la commutation d&#39;une cellule milieu de culture supplémenté avec 10% de sérum de veau foetal. Plus important encore, ces cellules souches tumorales différenciées en Tuj1 + ou + NeuN neurones, les astrocytes et GFAP + + CNPase oligodendrocytes, soulignant ainsi leur multi-puissance. En outre, ces cellules ont été capables de propager des médulloblastomes secondaire lorsque orthotopique transplantées chez des souris hôte.

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

Ce protocole décrit l&#39;isolement, l&#39;enrichissement et l&#39;entretien des cellules tumorales médulloblastome souches dérivées de souris mutantes avec ectopique Sonic activité de la voie Hedgehog.

L&#39;isolement, l&#39;enrichissement et l&#39;entretien des cellules souches Médulloblastome

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 vues.  Vanderbilt University. Ce protocole décrit l'isolement, l'enrichissement et l'entretien des cellules tumorales médulloblastome souches dérivées de souris mutantes avec ectopique Sonic activité de la voie Hedgehog.

Vidéo: 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.

Intracrânienne allogreffe orthotopique de cellules dans des souris immunodéprimées Médulloblastome

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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Neurosciences

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.

L&#39;isolement et expansion des cellules souches neurales de souris adultes utilisant le test Neurosphère

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.

Isolement et culture des cellules souches neurales Crête des follicules pileux humains

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.

Le test neuroblaste: Un test pour la génération et l&#39;enrichissement des cellules souches neuronales à partir de différenciation des cellules souches neurales Progeny Par cytométrie en flux

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.

Isolement et culture de cellules de crêtes neurales de tube neural embryonnaire murin

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.

Isolement, Culture et maintenance à long terme des mésencéphalique primaire dopaminergiques neurones à partir de cerveaux embryonnaires de rongeurs

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.

Isolement et culture des adultes cellules souches neurales de la Subcallosal Zone Souris

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.

Isolement et Culture des embryons de souris des cellules souches neurales

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 modèle évolutif pour étudier les effets des blessures contondantes chez le poisson-zèbre adulte

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 en tant qu’outil d’apprentissage associatif pour l’évaluation cognitive dans les études d’apprentissage et de mémoire à l’aide de poissons-zèbres adultes

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