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>Prepare agsNSC Basal Medium + 5% FBS by adding 5 mL FBS (LS-1012) to 95 mL agsNSC Basal Medium</li>
<li>Prepare agsNSC Expansion Medium by adding 500 &mu;L B-27 Supplement (Invitrogen) to 50 mL agsNSC Basal Medium. Divide the remainder of the B27 into 1mL aliquots and store at -20&deg;C until use.</li>
<li>Warm 25 mL of agsNSC Basal Medium + 5% FBS and 20 mL of agsNSC Expansion Medium in a 37&deg;C water bath.</li>
<li>Coat flasks used for expansion with poly-L-ornithine
<ol>
<li>Thaw the poly-L-ornithine (10 &mu;g/mL) working solution. Store the solution between 2-8&deg;C for up to one month once thawed.</li>
<li>Add 8 mL Poly-L-Ornithine working solution to one vented T-75 flask and evenly distribute the solution over the entire culture surface of the flask. (Adjust amount of working solution for other culture vessels that may be used. Use 0.5 - 1.0 mL Poly-L-Ornithine working solution per 10 cm2 culture surface.)</li>
<li>Incubate flasks at 37&deg;C for a minimum of 24 hours. (Use of an incubator without added CO2 is preferred for incubation of Poly-L-Ornithine coated culture vessels.) Coated flasks can be stored for up to two weeks in the incubator.</li>
<li>Prior to use, aspirate Poly-L-Ornithine working solution from the flask and wash twice with sterile tissue culture grade water. Aspirate liquid until the flask is nearly dry before use.</li>
</ol>
</li>
<li>Remove a vial from the Dewar and check that the vial cap is securely sealed. Hold the top of the vial leaving the bottom half of the vial in a 37&deg;C water bath for approximately 60-90 seconds or until vial is almost completely thawed; a small amount of ice should still be visible. To avoid potential contamination, keep the vial cap out of the water. Do not over thaw, as doing so may damage the cells.</li>
<li>Spray the exterior of the vial with 70% ethanol or isopropanol, then place the vial in a biological safety cabinet. Remove the cap carefully to avoid contamination or splatter.</li>
<li>Gently re-suspend the cells in the vial by adding 1 mL of agsNSC Basal media + 5% FBS using a sterile pipette. Transfer the re-suspended cells to 25 mL of warm agsNSC Basal Medium + 5% FBS in a 50 mL centrifuge tube.</li>
<li>Rinse the cryo-vial once with diluted cell suspension. Centrifuge the cells at 150 x g for 6 minutes. For best results, calculate the speed for each individual centrifuge type.</li>
<li>While the cells are spinning down, add 10 mL of agsNSC Expansion Medium to the dry poly-L-ornithine coated T-75 flask and add 40 &mu;L of rh FGF-basic (40ng/mL). Following centrifugation, aspirate the supernatant fluid taking care to not aspirate the pellet. Re-suspend the cells in 10 mL of agsNSC Expansion Medium. Transfer the re-suspended cells in agsNSC Expansion Medium into the media in the T-75 flask.</li>
<li>Gently rock the culture vessel side-to-side to evenly distribute cells within the vessel.</li>
<li>Place seeded culture vessel in a 37&deg;C, 5% CO2 incubator. Cells should attach to the flask and begin to form processes within two hours of seeding. If the cells remain floating and balled up within the medium, then centrifuge cells in 5% FBS and run steps 1.8-1.10 with fresh medium and flask.</li>
</ol> 

2. 	Expansion of AgsNSC
<ol>
<li>Two days (~48 hours) after thawing and seeding the cells, a full medium replacement should be performed using 20 mL of agsNSC Expansion Medium plus a 40 &mu;L spike of rh FGF-basic.</li>
<li>Spike flasks with 40 &mu;L of rh FGF-basic on the day following inoculation. The cells will be ready to trypsinize when they have reached 75-80% confluence by the third or fourth day after inoculation. Culturing the undifferentiated stem cells for longer than 4 days after inoculation is not recommended because longer incubation will allow cells to begin to differentiate to neurons and neurons may be injured by trypsinization during step 3. The rh FGF-basic can be stored at 4&deg;C for up to one week and still maintain activity. The rh FGF-basic can also go through one additional freeze/thaw at -20&deg;C if more time is needed.</li>
</ol>
3. 	Trypsinization of Cells
<ol>
<li>agsNSC should be passaged 3 to 4 days after inoculation. Prepare a poly-L-ornithine coated flask between 24 hrs and one week before passaging if the agsNSC will be expanded again.</li>
<li>Aspirate the medium from the culture vessel. Add 2.5 mL of 0.05% Trypsin/0.02% EDTA (CM-0017) to the vessel for each 75 cm2 of surface area. Swirl gently to ensure all cells are coated with the Trypsin/EDTA. Cells normally begin to detach from the surface within 60 seconds, depending on the confluence of the cells. Do not over trypsinize, as doing so will damage the cells. Gently tapping the culture vessel from several sides will encourage detachment.</li>
<li>Once the cells become detached, add 10 mL of agsNSC Basal Medium + 5% FBS to the flask. Gently swirl to ensure all of the trypsin solution is neutralized. Using aseptic laboratory techniques, pipette the cells into a sterile centrifuge tube containing 10 mL of agsNSC Basal medium +5% FBS. Repeat by washing the flask again with an additional 10 mL of agsNSC Basal Medium + 5% FBS and combine with the first wash.</li>
<li>Centrifuge cells at 150 x g for 6 minutes as described above. After centrifugation, the cells should form a clean loose pellet. Aspirate neutralized trypsin and medium from the centrifuge tube and re-suspend the cell pellet in 10 mL (per T-75 flask) of pre-warmed agsNSC Basal Medium + 5% FBS by gently pipetting up and down 2X with a 10 mL pipette. Perform a cell count as described below.</li>
</ol>
4. 	Standard Calculation for Plating of Cells
<ol>
<li>Using aseptic technique, swirl the cell suspension and transfer 15 &mu;L of the cell suspension to a microcentrifuge tube. Add 15 &mu;L of 0.4% Trypan Blue. Mix gently and load 10 &mu;L of cell suspension to each of the chambers of a clean hemacytometer such as a Bright Line Counting Chamber (catalog #1490: Hausser Scientific, <a href="http://www.hausserscientific.com/hausserbrightlinedirect.htm">http://www.hausserscientific.com/hausserbrightlinedirect.htm</a>).</li>
<li>Count a minimum of 4 quadrants on the hemacytometer. The blue cells that are positive for Trypan Blue are dead and should not be counted. For accurate cell counts, the optimal number of cells per quadrant should be 25-75 cells.</li>
<li>After counting the cells, calculate the average of the 4 quadrants. Multiply the cell count average by 10,000 and by the dilution factor (2 if using the recommended volumes) to determine the number of cells per mL. Note that if total percentage viability is less than 92% there may be a number of causes. One reason for low viability may be over trypsinization, prior to trypsin neutralization with FBS. Another reason may be that the concentration of serum (FBS) to trypsin is too low and the cells continue to be trypsinized. Thirdly, the cells may have been too confluent, causing stem cells to differentiate due to cell-cell contact, leading to injury during trypsinization. If this method is followed closely a yield of 3 or more doublings may be expected, giving a population of >4,000,000 cells from an initial number of ~500,000.</li>
</ol>
5. 	Inoculation of Another Expansion Flask
<ol>
<li>To determine the total number of cells required to inoculate one vessel, multiply the desired seeding density (6,600 cells per cm2 or 500,000 cells per T-75 flask) by the surface area (in cm2) of the vessel to be inoculated. If inoculating more than one vessel multiply the number of cells per vessel by the total number of vessels. To determine the volume (mL) of cell suspension needed to inoculate each vessel, divide the number of cells required to inoculate one vessel by the number of viable cells/mL in the cell suspension. If inoculating more than one vessel calculate the total volume of cell suspension needed by multiplying the volume needed for one vessel by the total number of vessels to be inoculated.</li>
<li>Centrifuge the desired volume of cells in agsNSC Basal Medium + 5% FBS, leaving a loose pellet. Normally 500,000 cells are brought to a total volume of 25 mL in agsNSC Basal Medium +5%FBS as described previously. Aspirate liquid and re-suspend the loose pellet in 10 mL of agsNSC Expansion Medium This extra centrifugation step is required to remove FBS before inoculation. Add 10 mL of pre-warmed agsNSC Expansion Medium to the T-75 flask. Add the 10 mL of resuspended cell suspension to the T-75 flask then add 40 &mu;L of rh FGF-basic to the flask. Consider that the trypsinization and inoculation should take no longer than 1.5 hrs when deciding the maximum number of flasks to work with at any one time. One should also have enough media for complete media changes if using numerous flasks.</li>
<li>Gently rock the flask from side to side to evenly distribute the cells and place the culture vessels into a 37&deg;C, 5% CO2 incubator, as described previously.</li>
</ol>
6. 	Dilution of Cells for Inoculation of the Well Plates for Differentiation
<ol>
<li>Any multi-well format might be used, but inoculation into BD Biocoat Poly-L-lysine coated 96 well plates is described here. Determine the volume of cell suspension needed for your experiment by multiplying the number of wells to be inoculated by 0.2 mL per well for a 96 multi-well plate and add approximately 10% to cover any loss in volume during the inoculation. Calculate the dilution to a cell density of 75,000 cells/mL. Determine the amount of FBS to be added to the Differentiation Medium to achieve a final concentration of 2% FBS (Table 1). Mix the FBS and Differentiation Medium. Carefully mix the cells with the calculated volume of Differentiation Medium with FBS. (note: During centrifugation the cells are in agsNSC Basal Medium + 5% FBS. While FBS protects cells during centrifugation and facilitates cell adhesion FBS inhibits differention. Diluting FBS to 2% will allow the adhesion of the cells to the multi-well surface. The 2% FBS is then diluted further to promote differentiation. Insulin-Transferrin-Selenium(ITS-e) is also included in the Differentiation Medium to promote differentiation.</li>
<li>In a biosafety cabinet, dispense 200 uL of diluted cell suspension from a sterile combitip reservoir to each well of a Poly-L-lysine coated 96-well plate using a multi-channel pipette and large orifice tips. Incubate the plates for 1.5-2 hours at 37&deg;C, 5% CO2. After the cells have attached, after about 90 to 120 minutes, carefully remove 50% (100 &mu;L for 96-well plates) of the medium from each well using large orifice pipette tips. Carefully replace the volume removed with warm agsNSC Differentiation Medium, again using large orifice pipette tips. (Use of standard orifice tips may injure the cells).</li>
<li>Two days after inoculation (~ 48 hours), perform a 50% medium change using agsNSC Differentiation Medium and large orifice pipette tips.</li>
</ol>
7. 	Maintenance of Neurons
<ol>
<li>Prepare Neuronal Maintenance Medium using NeuraLife, glutamine and B27 per the instructions. Neuronal Maintenance Medium should be prepared weekly. </li>
<li>Two days after the last agsNSC Differentiation Medium change, normally Day 4 post inoculation, perform a 50% medium replacement using Neuronal Maintenance Medium. </li>
<li>Continue to perform 50% medium replacements using Neuronal Maintenance Medium every 2 to 3 days. Neuronal processes should appear within a few days of changing to Neuronal Maintenance Medium. The cells should be used within 3 weeks of being transferred to Neuronal Maintenance Medium. Neurons can be identified using a number of commercially available neuronal markers. Astrocytes will also always be observed. </li>
<li>Culture populations typically express a greater than 40% ratio of neurons to total cell number. The use of neuron maintenance media other than NeuraLife may provide inferior cells that do not look morphologically mature and may not survive as long in culture.</li>
</ol>
8. Representative Results:
The Arctic ground squirrel adult neural stem cells will continue to double every 24 hrs for 3 to 5 days for at least two passages when seeded at 500,000-600,000 cells/75mm flask. These cells will easily differentiate with the removal of basicFGF and B27 and the presence of 1-2% FBS and 1% Insulin/Transferrin/Selenium, and will become TUJ1 (Covance) positive neurons within 14-21 days (see Figure 2). The ratio of neurons to total cells will range between 40-60%, dependent on the age of the culture (Figure 3). Neurons may be utilized for experimentation from 12 days post-seeding through at least 21 days post-seeding.

<img src="/files/ftp_upload/2199/2199fig1.jpg" alt="Figure 1" /> 
Figure 1. Photomicrograph of AGS adult hippocampal neural stem cells. 
Phase photomicrograph of AGS adult hippocampal neural stem cells following 3 days of growth in agsNSC basal media in Poly-L-Ornithine coated flasks. Doubling occurs approximately every 24hrs. Photo at 100X magnification.
<img src="/files/ftp_upload/2199/2199fig2.jpg" alt="Figure 2" /> 
Figure 2. Fluorescent micrograph of differentiated AGS adult hippocampal neural stem cells 
AGS neural stem cells were subjected to differentiation media for 4 days, then maintained in Neuralife for 17 more days prior to fixing. Cells were fixed with 4% paraformaldehyde and neurons identified using TUJ1 primary antibody (Covance) and Alexa Fluor 568 secondary antibody (green) from Invitrogen. Hoechst dye 33343 was added to stain all nuclei (blue). Cell number, neuron number and neuron ratio were determined using neurite outgrowth software and the Arrayscan instrument from Cellomics. This photo represents approximately 57% neurons. 10X magnification
<img src="/files/ftp_upload/2199/2199fig3.jpg" alt="Figure 3" /> 
Figure 3. Neuron numbers and Neuron Ratio of Differentiated AGS neural stem cells at three culture ages 
AGS neural stem cells were subjected to differentiation media for 4 days, then maintained in Neuralife for 12, 15 and 17 days prior to fixing. Cells were fixed and neurons identified with TUJ1 primary antibody (Covance). Hoechst dye was added to stain all nuclei for total cell identification. Cell number, neuron number and percent neuron ratio were determined using neurite outgrowth software and the Arrayscan instrument from Cellomics. Values represent mean of 15 wells per condition + SD.

Table 1. Sample calculations used in passaging and seeding
<ol>
<li>Using 700,000 cells/mL cell suspension</li>
<li>Using a cell density of 75,000 cells/mL or 15,000 cells per well of 0.2 mL volume in each well.</li>
<li>Inoculating 96 wells at 0.2 mL per well = 19.2 mL or desired final volume 21 mL.</li>
<li>(Final Concentration x Final Volume) &#247; Initial Concentration = Initial Volume 
	(21 mL x 75,000 cells/mL) &#247; 700,000 = 2.25 mL</li>
<li>Volume of FBS in cell suspension. 2.25 mL = 2.25 mL x 0.05 = 0.1125 mL</li>
<li>Volume of FBS needed in 21 mL = 21 mL x 0.02 = 0.42 mL</li>
<li>Volume of FBS to add to Differentiation Medium = 0.42 mL - 0.1125 mL = 0.3075 mL</li>
<li>Volume of Differentiation Medium = 21 mL - 2.25 mL cell suspension - 0.3075 mL 5%FBS= 18.44 mL.</li>
<li>Combine 18.44 mL Differentiation Medium with 0.308 mL FBS and add 2.25 mL of cell suspension.</li>
</ol>

<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>

J.A. Kelleher-Andersson is the founder of Neuronascent and is the inventor on the Arctic ground squirrel patent applications. Neuronascent receives royalties on the sale of AGS media and stem cells. R.C. McGee is employed by Lifeline, Inc. which distributes the media and AGS neural stem cells.

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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		<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 ...

growth-differentiation-adult-hippocampal-arctic-ground-squirrel

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9.5K Views.  University of Alaska at Fairbanks. 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.

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

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 chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

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

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

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

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

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

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

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

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

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

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

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

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

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

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

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

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

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

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...