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...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<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>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>
		</table>
		</div>

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 ...

Research

JoVE Journal

Neuroscience

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

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Utvidelse av embryonale og voksne nevrale stamceller ved<em&gt; In Utero</em&gt; Electroporation eller Viral stereotaksiske Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Isolering og kultur neural crest Celler fra Embryonic murin Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Promotion of Survival og Differensiering av nevrale stamceller med fibrin og vekstfaktor Cocktails etter alvorlig ryggmargsskade

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Økende nevrale stamceller fra konvensjonelle og ikke-konvensjonelle Regions of Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

En mus, to kulturer: Isolation and Culture of Adult nevrale stamceller fra de to nevrogene Soner Individuell Mus

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Enumeration of Neural Stem Cells Using Clonal Assays

Opplisting av Neural stamceller Bruke Klonale Analyser

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Isolasjon og Culture of Adult Neural stamceller fra mus Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Utvikling av en raffinert protokoll for Trans-Innbukking Subretinal transplantasjon av menneskelig netthinnens Pigment epitelceller i rotte øyne

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Isolasjon og kultur av embryonale mus embryonale stamceller

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 ...