Name: isolation, enrichment, and maintenance of medulloblastoma stem cells
Uploaded: 2010-09-01T17:00:00
Description: Watch this Scientific Journal Video about Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells at JoVE.com

Это исследование было поддержано грантами Вандербильт-Инграм онкологический центр поддержки Грант (P30 CA068485), опухоли мозга у детей и Фонд Национальных институтов здравоохранения (NS042205).

1. Микро-рассечение опухоль мозжечка, Диссоциация опухолевой ткани и покрытие <ol><li> Восстановление опухолевой ткани <ol><li> Больных мышей медуллобластомой часто runted, отображается гидроцефалия и типичных неврологических симптомов, включая паралич задних и неудачи, чтобы восстановить по...

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

Мы описываем эффективный способ изолировать, обогащать и поддерживать медуллобластомой стволовых клеток из первичной опухоли ткани извлекаются из мутантных мышей с конститутивно Ежик сигнализации. Мы обнаружили, что один важный шаг в создании успешно здоровых стволовых клеток опух?...

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		<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

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

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

Этот протокол описывает изоляции, обогащение, и поддержание медуллобластомой опухоли стволовые клетки, полученные из мутантных мышей с внематочной Соник деятельности путем ежа.

Изоляция, обогащению и техническому обслуживанию ячеек медуллобластомой Стволовой

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

Research

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Neuroscience

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Внутричерепное Ортотопическая аллотрансплантации медуллобластомы Ячейки в ослабленной иммунной системой Мыши

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Выделение и расширение взрослой мыши нервные стволовые клетки Использование Neurosphere Пробирной

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-ячейки изображения сенсорных клеток-предшественников органов в интактных Drosophila Куколки

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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

Выделение и культуры нервного гребня стволовых клеток из человеческих волоса

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

Нейробластов Анализ: Анализ для создания и обогащения нейронов клетки-предшественники из Дифференцируя нейронных потомков стволовых клеток методом проточной цитометрии

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

Выделение и культуры клеток нервного гребня из эмбриональных мышей нервной трубки

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

Изоляция, культуры и долгосрочного поддержания первичного теменной дофаминергических нейронов из эмбриональных грызунов Brains

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

Выделение и культуры взрослых нервные стволовые клетки от мыши зоны расположенный под мозолистым телом

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

Изоляции и культуры нервных стволовых клеток эмбриона мыши

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 ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Определение клеточных поверхностных маркеров первичных нейронных стволовых и клеток-прародителей путем метаболической маркировки сиалогликан

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...