Name: es cell-derived neuroepithelial cell cultures
Uploaded: 2006-11-30T00:00:00
Description: Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

Paso 1 <ol><li> Placa de mitomicina-C ggrowth inhibido (10 mg / ml durante 2,5 horas) MS5 las células a una densidad de 70.000 / cm2 en recubiertos de gelatina (0,01% durante 30 minutos) 6 placas en α-MEM medios de comunicación. </li><li> Cuando las células se unen y se han formado una monocapa (sobre el crecimiento de la noche), cambiar a SRM. </li><li> Manualmente aislar colonias de células ES de los cultivos de células ES mediante una jeringa con una aguja de 27 G ½. </li><li> Tritrurate las colonias con cuid...

<ol>
	<li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28, 31-40 (2000).</li><li>Perrier, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proc Natl Acad Sci U S A. 101, 12543-12548 (2004).</li><li>Pruszak, J., Isacson, O., Sullivan, S., Cowan, C., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+into+dopaminergic+neurons.">Directed differentiation of human embryonic stem cells into dopaminergic neurons.</a> Human Embryonic Stem Cells: The Practical Handbook. , (2007).</li><li>Pruszak, J., Sonntag, K. C., Aung, M. H., Sanchez-Pernaute, R., Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+and+methods+for+cell+sorting+of+human+embryonic+stem+cell-derived+neural+cell+populations.">Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations.</a> Stem Cells. 25, 2257-2268 (2007).</li><li>Sonntag, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+yield+of+neuroepithelial+precursors+and+midbrain-like+dopaminergic+neurons+from+human+embryonic+stem+cells+using+the+bone+morphogenic+protein+antagonist+noggin.">Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin.</a> Stem Cells. 25, 411-418 (2007).</li><li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dopaminergic+neurons+and+pigmented+epithelia+from+primate+ES+cells+by+stromal+cell-derived+inducing+activity.">Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity.</a> Proc Natl Acad Sci U S A. 99, 1580-1585 (2002).</li><li>Ko, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+neural+precursors+as+a+continuous,+stable,+and+on-demand+source+for+human+dopamine+neurons.">Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons.</a> J Neurochem. 103, 1417-1429 (2007).</li><li>Hong, S., Kang, U. J., Isacson, O., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+precursors+derived+from+human+embryonic+stem+cells+maintain+long-term+proliferation+without+losing+the+potential+to+differentiate+into+all+three+neural+lineages,+including+dopaminergic+neurons.">Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons.</a> J Neurochem. , (2007).</li><li>Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+subtype+specification+from+embryonic+stem+cells.">Neural subtype specification from embryonic stem cells.</a> Brain Pathol. 16, 132-142 (2006).</li><li>Pankratz, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neural+differentiation+of+human+embryonic+stem+cells+via+an+obligated+primitive+anterior+stage.">Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage.</a> Stem Cells. 25, 1511-1520 (2007).</li><li>Lazzari, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+derivation+of+neural+rosettes+from+cloned+bovine+blastocysts:+a+model+of+early+neurulation+events+and+neural+crest+specification+in+vitro.">Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro.</a> Stem Cells. 24, 2514-2521 (2006).</li><li>Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B., Goldstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+peripheral+sensory+and+sympathetic+neurons+and+neural+crest+cells+from+human+embryonic+stem+cells.">Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells.</a> Stem Cells. 23, 923-930 (2005).</li></ol>

Este protocolo demuestra los diferentes pasos en la creación y aislamiento de células neuroepiteliales de células madre embrionarias humanas con SDIA. La aplicación de este método es múltiple y se ha utilizado en muchos protocolos para producir neuronas específicas (por ejemplo 1, 2, 5-9). Las rosetas se cree que se asemejan a las células del tubo neural con un fenotipo anterior (2, 5, 10) y también contienen células progenitoras de la cresta neural (11, 12). Además, conservan un cierto nivel de plasticidad, ya que pueden ser mod...

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		<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>L-glutamine</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>alpha-MEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>12571</td>
<td>&nbsp;</td>
<tr>
<td>penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Knockout-DMEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>10829</td>
<td>&nbsp;</td>
<tr>
<td>Knockout serum replacement</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>10828</td>
<td>&nbsp;</td>
<tr>
<td>MEM non-essential amino acid solution</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>12383</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11330</td>
<td>&nbsp;</td>
<tr>
<td>N2-A supplement</td>
<td>&nbsp;</td>
<td>Stem Cell Technologies</td>
<td>07152</td>
<td>&nbsp;</td>
<tr>
<td>mitomycin-C</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M0503</td>
<td>&nbsp;</td>
<tr>
<td>gelatine type-A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-ornithine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4957</td>
<td>0.01 % solution </td>
</tr>
<tr>
<td>laminin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-2020</td>
<td>&nbsp;</td>
<tr>
<td>fibronectin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F2006</td>
<td>&nbsp;</td>
<tr>
<td>basic fibroblast growth factor (bFGF)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13256</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe with 27 1/2 G needle</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>309623</td>
<td>&nbsp;</td>
<tr>
<td>N2-A media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>DMEM/F12 + 1% N2-A supplement </td>
</tr>
<tr>
<td>Serum replacement media (SRM)</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Knockout-DMEM + 20 % Knockout serum replacement +1% MEM non-essential amino acid solution + 2 mM L-glutamine</td>
</tr>
<tr>
<td>&#x03B1;-MEM media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&#x03B1;-MEM + 10 % FBS + 2 mM L-glutamine + 1%penicillin/streptomycin</td>
</tr>
<tr>
<td>MS5</td>
<td>cell line</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>stromal cells </td>
</tr>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for tissue culture</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

es cell-derived neuroepithelial cell cultures

Células madre embrionarias tienen el potencial de diferenciarse en células de todas las capas germinales, que los convierte en una herramienta atractiva para el desarrollo de nuevas terapias. En general, la diferenciación de las células madre embrionarias sigue el concepto de generar primero las células progenitoras inmaduras, lo que puede ser propagado y diferenciarse en fenotipos celulares maduros. Esto también se aplica ES células derivadas de la neurogénesis, en el que el desarrollo de las células nerviosas después de dos pasos principales: en primer lugar, la derivación y la expansión de inmaduros precursores neuroepiteliales y la segunda, su diferenciación en células maduras neural. Un método común para producir progenitores neurales de células madre embrionarias se basa en el cuerpo embrioides (EB) la formación, lo que revela la diferenciación de las células de todas las capas germinales incluyendo neuroectodermo. Un método alternativo y más eficiente para inducir el desarrollo de células neuroepiteliales utiliza células estromales derivadas de la actividad de la inducción (SDIA), que se puede lograr mediante el cultivo de células madre embrionarias cooperación con los huesos del cráneo derivadas de la médula las células del estroma (1). Tanto, la formación de EB y SDIA, revelan el desarrollo de la roseta-como las estructuras, que se cree que se asemejan en el tubo neural y / o progenitores neurales como la cresta. Los precursores neuronales pueden ser aislados, ampliado y diferenciado aún más hasta convertirse en neuronas específicas y las células gliales con las condiciones definidas por la cultura. Aquí se describe la generación y el aislamiento de rosetas como en los experimentos de co-cultivo con la línea de células del estroma MS5 (2-5).

Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

ES Cell-derived Neuroepithelial Cell Cultures

Derivación de los precursores neuroepiteliales de células madre embrionarias (ES) las células utilizando células estromales derivadas de la actividad de inducción (SDIA).

ES células derivadas de células de las Culturas neuroepiteliales

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

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Freezing Human ES Cells

Congelar las células madre embrionarias humanas

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

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Human ES cells: Starting Culture from Frozen Cells

Células madre embrionarias humanas: Cultura a partir de células congeladas

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Propagation of Human Embryonic Stem (ES) Cells

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EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

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Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

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The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

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Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...