Name: growth and differentiation of adult hippocampal arctic ground squirrel neural stem cells
Uploaded: 2011-01-07T16:00:00
Description: Watch this Scientific Journal Video about Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells at JoVE.com

This work was supported by US Army Medical Research and Materiel Command grant # 05178001 and by NS041069-06 from The National Institute of Neurological Disorders and Stroke. We thank Joel Vonnahme for helpful comments on the protocol.

1. 	AGS Neural Stem Cell and Media Preparation
<ol>
<li>Adult hippocampal Arctic ground squirrel (AGS) neural stem cells isolated in a manner previously decribed9 can now be obtained commercially as cryopreserved vials. Cells are shipped overnight in insulated packages containing dry ice to ensure that the cells remain in a cryopreserved state. To maintain integrity, unpack the cells immediately upon receipt and store at lower than -150&deg;C, or in the vapor phase of a liquid nitrogen Dewar.</li>
<li>Pr...

<ol>
	<li>Barnes, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze+avoidance+in+a+mammal:+body+temperatures+below+0+degree+C+in+an+Arctic+hibernator.">Freeze avoidance in a mammal: body temperatures below 0 degree C in an Arctic hibernator.</a> Science. 244, 1593-1595 (1989).</li><li>Dave, K. R., Prado, R., Raval, A. P., Drew, K. L., Perez-Pinzon, 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=The+Arctic+ground+squirrel+brain+is+resistant+to+injury+from+cardiac+arrest+during+euthermia.">The Arctic ground squirrel brain is resistant to injury from cardiac arrest during euthermia.</a> Stroke. 37, 1261-1265 (2006).</li><li>Dave, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+epsilon+activation+delays+neuronal+depolarization+during+cardiac+arrest+in+the+euthermic+arctic+ground+squirrel.">Protein kinase C epsilon activation delays neuronal depolarization during cardiac arrest in the euthermic arctic ground squirrel.</a> J Neurochem. 110, 1170-1179 (2009).</li><li>Ross, A. P., Christian, S. L., Zhao, H. W., Drew, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+tolerance+to+oxygen+and+nutrient+deprivation+and+N-methyl-D-aspartate+in+cultured+hippocampal+slices+from+hibernating+Arctic+ground+squirrel.">Persistent tolerance to oxygen and nutrient deprivation and N-methyl-D-aspartate in cultured hippocampal slices from hibernating Arctic ground squirrel.</a> J Cereb Blood Flow Metab. 26, 1148-1156 (2006).</li><li>Christian, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arctic+ground+squirrel+(Spermophilus+parryii)+hippocampal+neurons+tolerate+prolonged+oxygen-glucose+deprivation+and+maintain+baseline+ERK1/2+and+JNK+activation+despite+drastic+ATP+loss.">Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen-glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss.</a> J Cereb Blood Flow Metab. 28, 1307-1319 (2008).</li><li>Jin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+stroke-induced+neurogenesis+in+the+human+brain.">Evidence for stroke-induced neurogenesis in the human brain.</a> Proc Natl Acad Sci U S A. 103, 13198-13202 (2006).</li><li>Maysami, S., Lan, J. Q., Minami, M., Simon, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferating+progenitor+cells:+a+required+cellular+element+for+induction+of+ischemic+tolerance+in+the+brain.">Proliferating progenitor cells: a required cellular element for induction of ischemic tolerance in the brain.</a> J Cereb Blood Flow Metab. 28, 1104-1113 (2008).</li><li>Sung, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia/reoxygenation+stimulates+proliferation+through+PKC-dependent+activation+of+ERK+and+Akt+in+mouse+neural+progenitor+cells.">Hypoxia/reoxygenation stimulates proliferation through PKC-dependent activation of ERK and Akt in mouse neural progenitor cells.</a> Neurochem Res. 32, 1932-1939 (2007).</li><li>Brewer, G. 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+and+culture+of+adult+rat+hippocampal+neurons.">Isolation and culture of adult rat hippocampal neurons.</a> J Neurosci Methods. 71, 143-155 (1997).</li><li>Palmer, T. D., Takahashi, J., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+rat+hippocampus+contains+primordial+neural+stem+cells.">The adult rat hippocampus contains primordial neural stem cells.</a> Mol Cell Neurosci. 8, 389-404 (1997).</li></ol>

AGS adult neural stem cells are robust and can be expanded for numerous passages making them an excellent source of neural stem cells for study of basic neural stem cell properties. The expansion rate is approximately one doubling every twenty-four hours, but these cells must be passaged prior to reaching confluence because contact between cells will favor differentiation to astrocytes. Contact-mediated differentiation will therefore cause the ratio of neurons to total cell number to drop dramatically, if differentiati...

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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>agsNSC</td>
<td>&nbsp;</td>
<td>Lifeline</td>
<td>FC-0004</td>
<td>&nbsp;</td>
<tr>
<td>agsNSC Expansion kit</td>
<td>&nbsp;</td>
<td>Lifeline</td>
<td>LL-0008</td>
<td>Alternative source for DMEM/F12, B27 and rhFGFb (<a href="http://www.invitrogen.com">www.invitrogen.com</a>)</td>
</tr>
<tr>
<td>agsNSC differentiation kit</td>
<td>&nbsp;</td>
<td>Lifeline</td>
<td>LL-0009</td>
<td>Alternative source for DMEM/F12 and ITS-x (<a href="http://www.invitrogen.com">www.invitrogen.com</a>)</td>
</tr>
<tr>
<td>NeuraLife maintenance media kit</td>
<td>&nbsp;</td>
<td>Lifeline</td>
<td>LL-0012</td>
<td>Alternative sources are NeuraBasal (LifeLine) or Neurobasal (in vitrogen), glutamine and B27 (<a href="http://www.invitrogen.com">www.invitrogen.com</a>)Differentiated cells are maintained for up to 3 weeks in NeuroLife, but no more than 2 weeks in Neurobasal (<a href="http://www.invitrogen.com">www.invitrogen.com</a>)</td>
</tr>
<tr>
<td>Micrometer (such as a Bright Line Counting Chamber)</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>1490</td>
<td><a href="http://www.hausserscientific.com/hausserbrightlinedirect.htm">http://www.hausserscientific.com/hausserbrightlinedirect.htm</a></td>
</tr>
<tr>
<td>Biocoat Poly-L-Lysine coated 96 well plates</td>
<td>&nbsp;</td>
<td>BDBioscience</td>
<td>356516</td>
<td><a href="http://www.bdbiosciences.com">www.bdbiosciences.com</a></td>
</tr>
<tr>
<td>Large orifice pipette tips</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>02-707-141</td>
<td>Avoids neuronal cell damage when pippetting. <a href="http://www.fishersci.com">http://www.fishersci.com</a></td>
</tr>		</tbody>
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growth and differentiation of adult hippocampal arctic ground squirrel neural stem cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Watch this Scientific Journal Video about Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells at JoVE.com

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

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