The authors would like to acknowledge support from Spinal Cord Injury Ontario, the Ontario-China Research and Innovation Fund, the Toronto General and Western Hospital Foundation, and Physicians&#8217; Services Incorporated Foundation. The authors thank Dr. Tasneem Zahir for expert advice in human spinal cord NSPC culture, and Drs. Cindi Morshead and Iris Kulbatski for their expert advice in rat spinal cord NSPC isolation.

All animal procedures are approved by the Animal Care Committee of the University Health Network, Toronto ON Canada, in accordance with the policies established in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care. For the harvesting of human spinal cord tissue, approval was obtained from the University Health Network Research Ethics Board and from the Trillium-Gift of Life Foundation which oversees organ donation in Ontario, Canada.
1. Prepara...

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	<li>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=Mammalian+neural+stem+cells.">Mammalian neural stem cells.</a> Science. 287, 1433-1438 (2000).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Gritti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotential+stem+cells+from+the+adult+mouse+brain+proliferate+and+self-renew+in+response+to+basic+fibroblast+growth+factor.">Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.</a> J Neurosci. 16, 1091-1100 (1996).</li><li>Morshead, C. M., et al. <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+in+the+adult+mammalian+forebrain:+a+relatively+quiescent+subpopulation+of+subependymal+cells.">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> Neuron. 13, 1071-1082 (1994).</li><li>Weiss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+CNS+stem+cells+are+present+in+the+adult+mammalian+spinal+cord+and+ventricular+neuroaxis.">Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis.</a> J Neurosci. 16, 7599-7609 (1996).</li><li>Martens, D. J., Seaberg, R. M., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+infusions+of+exogenous+growth+factors+into+the+fourth+ventricle+of+the+adult+mouse+brain+increase+the+proliferation+of+neural+progenitors+around+the+fourth+ventricle+and+the+central+canal+of+the+spinal+cord.">In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord.</a> Eur J Neurosci. 16, 1045-1057 (2002).</li><li>Kulbatski, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocytes+and+radial+glia+derived+from+adult+rat+spinal+cord+progenitors:+morphological+and+immunocytochemical+characterization.">Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization.</a> J Histochem Cytochem. 55, 209-222 (2007).</li><li>Akesson, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+and+neuronal+survival+after+intraspinal+transplantation+of+human+spinal+cord-derived+neurospheres.">Long-term culture and neuronal survival after intraspinal transplantation of human spinal cord-derived neurospheres.</a> Physiol Behav. 92, 60-66 (2007).</li><li>Dromard, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+spinal+cord+harbors+neural+precursor+cells+that+generate+neurons+and+glial+cells+in+vitro.">Adult human spinal cord harbors neural precursor cells that generate neurons and glial cells in vitro.</a> J Neurosci Res. 86, 1916-1926 (2008).</li><li>Mothe, A. J., Zahir, T., Santaguida, C., Cook, D., Tator, C. H. <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/progenitor+cells+from+the+adult+human+spinal+cord+are+multipotent+and+self-renewing+and+differentiate+after+transplantation.">Neural stem/progenitor cells from the adult human spinal cord are multipotent and self-renewing and differentiate after transplantation.</a> PLoS One. 6, e27079 (2011).</li><li>Huettner, J. E., Baughman, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+identified+neurons+from+the+visual+cortex+of+postnatal+rats.">Primary culture of identified neurons from the visual cortex of postnatal rats.</a> J Neurosci. 6, 3044-3060 (1986).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <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+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> J Vis Exp. , (2010).</li><li>Reynolds, B. A., Rietze, R. L. <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+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Singec, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nat Methods. 3, 801-806 (2006).</li><li>Louis, S. A., Azari, H., Sharififar, S., Vedam-Mai, V., Reynolds, B. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+neural+stem+and+progenitor+cells+in+the+neural+colony-forming+cell+assay.">Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.</a> Stem Cells. 26, 988-996 (2008).</li><li>Conti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niche-independent+symmetrical+self-renewal+of+a+mammalian+tissue+stem+cell.">Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.</a> PLoS Biol. 3, e10 (2005).</li><li>Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+neural+stem+(NS)+cells+from+fetal+and+adult+forebrain.">Adherent neural stem (NS) cells from fetal and adult forebrain.</a> Cereb Cortex. 16, i112-i120 (2006).</li></ol>

During the dissection of the rat spinal cord tissue care should be taken not to damage the spinal cord while performing the laminectomy. It is easier to isolate the periventricular tissue when the spinal cord segments are intact. The tissue segments should be fully immersed in dissection buffer and the overlying meninges and white matter can be cut away as longitudinal strips with microscissors. Alternatively, fine tissue forceps can be used to peel the white matter away.
For the isolation pro...

NSPCs are multipotent cells committed to the neural lineage that can self renew and readily expand in vitro. We refer to these cells as a mixed population of neural stem/progenitor cells since they display properties of self-renewing multipotent stem cells and more restricted progenitors. NSPCs are found in both the fetal and adult brain and spinal cord1,2. In the adult, NSPCs are normally quiescent and reside within specific niches including the subventricular zone lining the lateral ventricles2-4, and the periventricular region surrounding the central canal of the spinal cord5,6.
Typically, NSPCs are cultured as free-floating neurospheres or as adherent monolayers in serum-free medium supplemented with EGF and bFGF, mitogens which select for the stem/progenitor cell populations. The neurosphere assay originally developed by Reynolds and Weiss2, is most commonly used to culture and expand neural stem cells. NSPCs show multipotency when they are plated in growth factor-free medium containing serum, differentiating into neurons, astrocytes, and oligodendrocytes. Multipotent, self-renewing NSPCs can be isolated and cultured from the adult rodent spinal cord when the cultured tissue includes regions of the central canal6,7. A potential advantage in using NSPCs generated from the adult spinal cord as opposed to generating cells from other regions is that these tissue-specific cells most closely resemble cells in the spinal cord that are lost or damaged following injury or disease.
Previous work showed that neurospheres derived from the adult human spinal cord could not be propagated long-term or passaged to generate sufficient numbers of cells8,9. However, with modifications in culturing conditions, we reported the expansion and transplantation of adult human spinal cord-derived NSPCs, demonstrating that self-renewing and multipotent NSPCs can be isolated from the adult human spinal cord of organ transplant donors10. Primarily, the removal of most of the white matter during dissection and culturing these cells on an adherent substrate in growth factor-enriched medium selected for proliferating adult human spinal cord NSPCs. In this protocol we describe the harvesting of the spinal cord from adult rat and from human organ transplant donors, dissection of the periventricular tissue, and the isolation, culture and expansion of NSPCs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1X PBS</td>
			<td>Life Technologies</td>
			<td>#10010023</td>
			<td>Dissection buffer</td>
		</tr>
		<tr>
			<td>1X HBSS</td>
			<td>Life Technologies</td>
			<td>#14175095</td>
			<td>Dissection buffer</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td># G-6152</td>
			<td>Prepare 30% glucose stock solution for dissection buffer and hormone mix</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>#15140-148</td>
			<td>Dissection buffer and culture medium</td>
		</tr>
		<tr>
			<td>Neurobasal-A&#160;</td>
			<td>Life Technologies</td>
			<td>#10888-022</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>L-glutamine, 200mM</td>
			<td>Life Technologies</td>
			<td>#25030-081</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Life Technologies</td>
			<td>#12587010</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>#11885084</td>
			<td>Hormone mix</td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Life Technologies</td>
			<td>#21700-075</td>
			<td>Hormone mix</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td># S-5761</td>
			<td>Prepare 7.5% NaHCO3 stock solution for hormone mix</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>#H9136</td>
			<td>Prepare 1M HEPES stock solution for hormone mix</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>#I-5500</td>
			<td>Hormone mix</td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>#T-2252</td>
			<td>Hormone mix</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td># P7505</td>
			<td>Hormone mix</td>
		</tr>
		<tr>
			<td>Selenium</td>
			<td>Sigma</td>
			<td>#S-9133</td>
			<td>Hormone mix</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>#P-6149</td>
			<td>Hormone mix</td>
		</tr>
		<tr>
			<td>EGF, mouse</td>
			<td>Sigma</td>
			<td>#E4127</td>
			<td>Prepare 100 &#956;g/ml stocks in B27 and aliquot; EFH medium</td>
		</tr>
		<tr>
			<td>EGF, human recombinant</td>
			<td>Sigma</td>
			<td>#E9644</td>
			<td>Prepare 100 &#956;g/ml stocks in B27 and aliquot; EFH medium</td>
		</tr>
		<tr>
			<td>bFGF, human recombinant</td>
			<td>Sigma</td>
			<td>#F0291</td>
			<td>Prepare 100 &#956;g/ml stocks in B27 and aliquot; EFH medium&#160;</td>
		</tr>
		<tr>
			<td>Heparin, 10000U</td>
			<td>Sigma</td>
			<td>#H3149</td>
			<td>Prepare 27.3 mg/ml stocks in hormone mix and aliquot; EFH medium</td>
		</tr>
		<tr>
			<td>Papain dissociation kit</td>
			<td>Worthington Biochemicals</td>
			<td>#LK003150</td>
			<td>Contains EBSS, papain, DNase, ovomucoid protease inhibitor with BSA</td>
		</tr>
		<tr>
			<td>Sodium Pentobarbital</td>
			<td>Bimeda &#8211; MTC Animal Health Inc</td>
			<td>DIN 00141704</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Forceps: Addisons</td>
			<td>Fine Science Tools</td>
			<td>#11006-12&#160;</td>
			<td>Serrated standard tip; micro-tip also available</td>
		</tr>
		<tr>
			<td>Fine Forceps: Dumont #4</td>
			<td>Fine Science Tools</td>
			<td>#11241-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Fine Science Tools</td>
			<td>#15024-10</td>
			<td>Round-handled Vannas</td>
		</tr>
		<tr>
			<td>Rongeurs</td>
			<td>Bausch &#38; Lomb</td>
			<td>N1430</td>
			<td></td>
		</tr>
		<tr>
			<td>10mm Petri dishes&#160;</td>
			<td>Nunc</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 Culture flasks</td>
			<td>Nunc</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m nylon cell strainer</td>
			<td>VWR</td>
			<td>CA21008-949</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Nunc</td>
			<td>CA73520-906</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, growth factor reduced (100X)</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Thaw according to directions and freeze aliquots; use diluted at a ratio of 40 &#956;l Matrigel: 1 ml SFM</td>
		</tr>
	</tbody>
</table>

isolation of neural stem/progenitor cells from the periventricular region of the adult rat and human spinal cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

Adult rat spinal cord cells grown in suspension culture in EFH medium will form small neurospheres (colonies of undifferentiated cells) within 1 week of initial plating. In primary cultures, most of the cells plated will die and growth factor-responsive stem cells will proliferate and are selected for in the EFH medium. By passage 3, there will be numerous free-floating neurospheres about 100 &#181;m in diameter (Figure 2A). Neurospheres are round and phase-bright, and under high magnification, cilia-lik...

Watch this Scientific Journal Video about Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord at JoVE.com

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

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12.1K Views.  Toronto Western Research Institute and Krembil Neuroscience Center. The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors. 