Name: generation of neural stem cells from discarded human fetal cortical tissue
Uploaded: 2011-05-25T12:00:00
Description: Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

This work was supported in part by the National Institutes of Health: HD054347 and NS063997-01 to VLS. This work was also supported in part by the Empire State Stem Cell Fund through the New York State Department of Health Contract #C024324 to VLS. The opinions expressed here are solely those of the author and do not necessarily reflect those of the Empire State Stem Cell Board, the New York State Department of Health, or the State of New York. VLS is a Doris Duke Clinical Scientist Developmental Award Recipient. We also thank Professor Timothy Vartanian for his gift of Anti-O1, Anti-O4 antibodies.

1. Preparation of solutions and materials for dissection and maintenance of neural stem cell culture
<ol>
	<li>Prepare 100ml dissection medium (KNOCKOUT DMEM/F12, Invitogen) ahead of time and refrigerate.</li>
<li>Prepare100 ml culture medium (Stem Pro NSC SFM, Invitrogen) and keep at 37 &deg;C in a water bath.</li>
<li>Prepare cell-freezing medium (KNOCKOUT DMEM/F12+10%FBS+ 5%DMSO) for long term cryopreservation of cells.</li>
<li>If desired, prepare 4% paraformaldehyde (PFA) for tissue fixation.</li>
<li>Sterile, aut...

<ol>
	<li>Gage, F. H., Ray, J., Fisher, L. 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,+characterization,+and+use+of+stem+cells+from+the+CNS.">Isolation, characterization, and use of stem cells from the CNS.</a> Annu. Rev. Neurosci. 18, 159-192 (1995).</li><li>Vescovi, A. L., Snyder, 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=Establishment+and+properties+of+neural+stem+cell+clones:+plasticity+in+vitro+and+in+vivo.">Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo.</a> Brain Pathol. 9, 569-598 (1999).</li><li>Schwartz, P., Bryant, P., Fuja, T., Su, H., O'Dowd, D., Klassen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+neural+progenitor+cells+from+post-mortem+human+cortex.">Isolation and characterization of neural progenitor cells from post-mortem human cortex.</a> J Neurosci Res. 74, 838-851 (2003).</li><li>Martinez-Serrano, A., Rubio, F. J., Navarro, B., Bueno, C., Villa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+and+progenitor+cells:+in+vitro+and+in+vivo+properties,+and+potential+for+gene+therapy+and+cell+replacement+in+the+CNS.">Human neural stem and progenitor cells: in vitro and in vivo properties, and potential for gene therapy and cell replacement in the CNS.</a> Curr Gene Ther. 1, 279-299 (2001).</li><li>Rajan, P., Snyder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+their+manipulation.">Neural stem cells and their manipulation.</a> Methods Enzymol. 419, 23-52 (2006).</li><li>Ruiz-Lozano, P., Rajan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+as+in+vitro+models+of+disease.">Stem cells as in vitro models of disease.</a> Curr Stem Cell Res Ther. 2, 280-292 (2007).</li><li>Sheen, V., Ferland, R., Harney, M., Hill, R., Neal, J., Banham, A., Brown, P., Chenn, A., Corbo, J., Hecht, J., Folkerth, R., Walsh, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+proliferation+and+migration+in+human+Miller-Dieker+neural+precursors.">Impaired proliferation and migration in human Miller-Dieker neural precursors.</a> Ann Neurol. 60, 137-144 (2006).</li><li>Bahn, S., Mimmack, M., Ryan, M., Caldwell, M., Jauniaux, E., Starkey, M., Svendsen, C., Emson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+target+genes+of+the+neuron-restrictive+silencer+factor+in+neurospheres+derived+from+fetuses+with+Down's+syndrome:+a+gene+expression+study.">Neuronal target genes of the neuron-restrictive silencer factor in neurospheres derived from fetuses with Down's syndrome: a gene expression study.</a> Lancet. 359, 310-315 (2002).</li><li>Ferland, R. J., Batiz, L. F., Neal, J., Lian, G., Bundock, E., Lu, J., Hsiao, Y. C., Diamond, R., Mei, D., Banham, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+neural+progenitors+along+the+ventricular+and+subventricular+zones+in+periventricular+heterotopia.">Disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia.</a> Hum Mol Genet. 18, 497-516 (2009).</li><li>Esposito, G., Imitola, J., Lu, J., De Filippis, D., Scuderi, C., Ganesh, V. S., Folkerth, R., Hecht, J., Shin, S., Iuvone, T., Chesnut, J., Steardo, L., Sheen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+functional+profiling+of+human+Down+syndrome+neural+progenitors+implicates+S100B+and+aquaporin+4+in+cell+injury.">Genomic and functional profiling of human Down syndrome neural progenitors implicates S100B and aquaporin 4 in cell injury.</a> Hum Mol Genet. 17, 440-457 (2008).</li><li>Esposito, G., Scuderi, C., Lu, J., Savani, C., De Filippis, D., Iuvone, T., Steardo, L. J. r., Sheen, V., Steardo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S100B+induces+tau+protein+hyperphosphorylation+via+Dickopff-1+up-regulation+and+disrupts+the+Wnt+pathway+in+human+neural+stem+cells.">S100B induces tau protein hyperphosphorylation via Dickopff-1 up-regulation and disrupts the Wnt pathway in human neural stem cells.</a> J Cell Mol Med. 12, 914-927 (2008).</li><li>Flax, J. D., Aurora, S., Yang, C., Simonin, C., Wills, A. M., Billinghurst, L. L., Jendoubi, M., Sidman, R. L., Wolfe, J. H., Kim, S. U., Snyder, 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=Engraftable+human+neural+stem+cells+respond+to+developmental+cues,+replace+neurons,+and+express+foreign+genes.">Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.</a> Nat Biotechnol. 16, 1033-1039 (1998).</li><li>Fults, D., Pedone, C. A., Morse, H. G., Rose, J. W., McKay, R. D. <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+characterization+of+a+human+primitive+neuroectodermal+tumor+cell+line+from+the+cerebral+hemisphere.">Establishment and characterization of a human primitive neuroectodermal tumor cell line from the cerebral hemisphere.</a> J Neuropathol Exp Neurol. 51, 272-280 (1992).</li><li>Conti, L., Cattaneo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+systems:+physiological+players+or+in+vitro+entities?.">Neural stem cell systems: physiological players or in vitro entities?.</a> Nat Rev Neurosci. 11, 176-187 (2010).</li><li>Svendsen, C. N., ter Borg, M. G., Armstrong, R. J., Rosser, A. E., Chandran, S., Ostenfeld, T., Caldwell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+rapid+and+long+term+growth+of+human+neural+precursor+cells.">A new method for the rapid and long term growth of human neural precursor cells.</a> J Neurosci Methods. 85, 141-152 (1998).</li></ol>

There are various approaches toward culture fresh tissue and producing human cell lines. Historically, fresh tissue has been harvested and cultured immediately to generate various cell types in the central nervous system. This approach however is clearly limited by the number of samples that can be obtained- which in the case for human samples, is usually quite small. Given the minimal degree of manipulation, freshly cultured neural cells provide the most reliable experimental system by limiting potential artifacts from ...

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>KNOCKOUT DMEM/F12</td>
 <td>Invitrogen</td>
 <td>12660-012</td>
 <td>Dissociation medium</td>
 </tr>
 <tr>
 	<td>Stem Pro NSC SFM</td>
 <td>Invitrogen</td>
 <td>A10509-01</td>
 <td>Culture medium</td>
 </tr>
 <tr>
 	<td>Fetal Bovine Serum</td>
 <td>Invitrogen</td>
 <td>10091-148</td>
 <td>Frozen medium</td>
 </tr>
 <tr>
 	<td>Hanks solution (-Ca2+, -Mg2+)</td>
 <td>Invitrogen</td>
 <td>14175-095</td>
 <td>Dissociation medium</td>
 </tr>
 <tr>
 	<td>DMSO</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>Frozen medium</td>
 </tr>
 <tr>
 	<td>EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>431788</td>
 <td>Dissociation medium</td>
 </tr>
 <tr>
 	<td>Paraformaldehyde</td>
 <td>Sigma-Aldrich</td>
 <td>158127</td>
 <td>Fixation solution</td>
 </tr>
 <tr>
 	<td>bFGF</td>
 <td>R&amp;D</td>
 <td>234-FSE</td>
 <td>Differentiation medium</td>
 </tr>
 <tr>
 	<td>SHH</td>
 <td>R&amp;D</td>
 <td>1845-SH</td>
 <td>Differentiation medium</td>
 </tr>
 <tr>
 	<td>PDGF-AA</td>
 <td>R&amp;D</td>
 <td>221-AA</td>
 <td>Differentiation medium</td>
 </tr>
 <tr>
 	<td>B27</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td>Differentiation medium</td>
 </tr>
 <tr>
 	<td>Mouse Anti-MAP2</td>
 <td>Sigma-Aldrich</td>
 <td>M2320</td>
 <td>1:200</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-DCX</td>
 <td>Cell signaling </td>
 <td>4604s</td>
 <td>1:200</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-GFAP</td>
 <td>DAKO</td>
 <td>Z0334</td>
 <td>1:200</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-S100B</td>
 <td>DAKO</td>
 <td>Z0311</td>
 <td>1:200</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-O1</td>
 <td>gifts of Professor Timothy Vartanian*</td>
 <td>&nbsp;</td>
 <td>1:50</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-O4</td>
 <td>Gifts of Professor Timothy Vartanian*</td>
 <td>&nbsp;</td>
 <td>1:50</td>
 </tr>
 <tr>
 	<td>40&mu;m cell strainer </td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
 
 </tbody>
</table>
* Timothy Vartanian, MD, PhD, Department of Neurology and Neuroscience, Weill Cornell Medical College, New York, USA

generation of neural stem cells from discarded human fetal cortical tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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