Name: production of chick embryo extract for the cultivation of murine neural crest stem cells
Uploaded: 2010-11-27T17:00:00
Description: Watch this Scientific Journal Video about Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells at JoVE.com

The support and advice of Markus Pajtler and Robert Bohrer were invaluable for the visualization of this protocol.

The authors state that experiments on animals were performed in accordance with the European Communities Council Directive (86/609/EEC), following the Guidelines of the NIH regarding the care and use of animals for experimental procedures and the regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at the University of Duisburg-Essen (Germany).

Part 1: Setting Up (not Shown on Video)
PREPARING MATERIALS AND SOLUTIONS (prior to taking th...

<ol>
	<li>Bronner-Fraser, M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+analysis+reveals+multipotency+of+some+avian+neural+crest+cells.">Cell lineage analysis reveals multipotency of some avian neural crest cells.</a> Nature. 335, 161-164 (1988).</li><li>Stemple, D. L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+stem+cell+for+neurons+and+glia+from+the+mammalian+neural+crest.">Isolation of a stem cell for neurons and glia from the mammalian neural crest.</a> Cell. 71, 973-985 (1992).</li><li>Rao, M. S., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+and+controlled+in+vitro+differentiation+of+murine+multipotent+neural+crest+stem+cells.">Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.</a> J Neurobiol. 32, 722-746 (1997).</li><li>Paratore, C., Goerich, D. E., Suter, U., Wegner, M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+glial+fate+acquisition+of+neural+crest+cells+are+regulated+by+an+interplay+between+the+transcription+factor+Sox10+and+extrinsic+combinatorial+signaling.">Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling.</a> Cell. 128, 3949-3961 (2001).</li><li>Kim, J., Lo, L., Dormand, E., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX10+maintains+multipotency+and+inhibits+neuronal+differentiation+of+neural+crest+stem+cells.">SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells.</a> Neuron. 38, 17-31 (2003).</li><li>Anderson, D. J., Groves, A., Lo, L., Ma, Q., Rao, M., Shah, N. M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+determination+and+the+control+of+neuronal+identity+in+the+neural+crest.">Cell lineage determination and the control of neuronal identity in the neural crest.</a> Review Cold Spring Harb Symp Quant Biol. 62, 493-504 (1997).</li><li>Dorsky, R. I., Moon, R. T., Raible, 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=Environmental+signals+and+cell+fate+specification+in+premigratory+neural+crest.">Environmental signals and cell fate specification in premigratory neural crest.</a> Review Bioessays. 22, 708-716 (2000).</li><li>Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+lineage+specification+in+the+neural+crest.">Factors controlling lineage specification in the neural crest.</a> Int Rev Cytol. 197, 1-33 (2000).</li><li>Dupin, E., Real, C., Ledouarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+crest+stem+cells:+control+of+neural+crest+cell+fate+and+plasticity+by+endothelin-3.">The neural crest stem cells: control of neural crest cell fate and plasticity by endothelin-3.</a> Int Rev Cytol. 73, 533-545 (2001).</li><li>Maurer, J., Fuchs, S., J&#228;ger, R., Kurz, B., Sommer, L., Schorle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+controlled+differentiation+of+neural+crest+stem+cell+lines+using+conditional+transgenesis.">Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis.</a> Differentiation. 75, 890-891 (2007).</li><li>Wu, Y. Y., Mujtaba, T., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+stem+and+precursor+cells+from+fetal+tissue.">Isolation of stem and precursor cells from fetal tissue.</a> Methods Mol Biol. 198, 29-40 (2002).</li></ol>

This protocol is based on modifications of procedures that have been described in the past10,11. In addition to former publications we here show the individual different steps of the process more expanded. This supports the experimenter with important details assuring correct proceedings and reliable outcomes. Further, as the dissection process is an essential part of CEE extraction we describe the procedure of taking the embryos out of the yolk sack in every detail. There are several important points to note ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>11 days incubated chicken eggs</td>
<td>&nbsp;</td>
<td>S&ouml;rries-Trockels Vermehrungszucht GmbH &amp; Co.KG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Alcoholic disinfection spray</td>
<td>&nbsp;</td>
<td>Sch&uuml;lke &amp; Mayr GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEMGlutamax</td>
<td>&nbsp;</td>
<td>GIBCO Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Syringe sterile (60 ml)</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tubes sterile (50; 15 ml)</td>
<td>&nbsp;</td>
<td>Greiner Bio-One GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipet tips sterile</td>
<td>&nbsp;</td>
<td>Starlab GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase typeIV-S</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Milipore stericups sterile (0.45; 0.22 &mu;l)</td>
<td>&nbsp;</td>
<td>Fisher Scientific AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipettes sterile (25; 10; 5 ml)</td>
<td>&nbsp;</td>
<td>Greiner Bio-One GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge L5-50</td>
<td>&nbsp;</td>
<td>Rotor type Ti-45</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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production of chick embryo extract for the cultivation of murine neural crest stem cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

Watch this Scientific Journal Video about Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells at JoVE.com

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

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