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. Preparation of Dissection Buffers and Culture Media
<ol>
	<li>For the isolation of rat spinal cord, prepare 100 ml dissection buffer (1x PBS + 0.6% glucose + 2% penicillin-streptomycin) and refrigerate. For human spinal cord prepare 100 ml dissection buffer (1x HBSS + 0.6% glucose + 2% penicillin-streptomycin) and refrigerate.</li>
	<li>Prepare 100 ml serum-free medium (SFM) and warm at 37 &#176;C. To prepare SFM, add 2 mM L-glutamine, 100 &#181;g/ml penicillin-streptomycin, 2% B27, and 10% hormone mix to Neurobasal-A medium. To prepare hormone mix, make a 1 : 1 DMEM/F12 medium containing 0.6% glucose, 3 mM NaHCO3, 5 mM HEPES, 25 &#181;g/ml insulin, 100 &#181;g/ml apo-transferrin, 10 &#181;M putrescine, 30 nM selenium, and 20 nM progesterone.</li>
	<li>Prepare the EFH growth factor-enriched plating media (to SFM add 20 ng/ml EGF, 20 ng/ml bFGF, and 2 &#181;g/ml heparin) and warm at 37 &#176;C. Ensure that human EFH contains human recombinant growth factors (see Table of Materials).</li>
</ol>
2. Harvesting and Dissection of Adult Rat Periventricular Spinal Cord Tissue
<ol>
	<li>Sterilize dissecting instruments in ethanol and rinse in sterile saline, or autoclave instruments in advance. Give rat (6 - 8 weeks old) a lethal injection of sodium pentobarbital (1 cc at 65 mg/ml) or overdose of anesthetic (i.e., 4% isofluorane) according to one&#8217;s institution approved animal protocol. Douse the back of the rat with 70% ethanol and remove the skin on the dorsal surface with large dissection scissors.</li>
	<li>Holding the scissors perpendicular to the dorsal surface, transversely cut the vertebral column above the hindlimbs. Use smaller scissors to longitudinally cut the dorsal muscle overlying the vertebral column in a rostral direction to expose the spinous processes.</li>
	<li>Starting at the exposed caudal end, insert rongeurs or a small blunt bone-cutting instrument extradurally into the lateral aspect of the spinal canal in the space between the spinal cord and the vertebral column. Make small cuts into the lamina (bony arches left and right of the spinous processes on each side of the vertebrae) and carefully peel away the lamina to expose the spinal cord (Figure 1A-D). Ensure the angle of the blades is shallow and parallel to the cord so the underlying spinal cord is not damaged.</li>
	<li>Continue laminectomy in the rostral direction to expose the thoracic and cervical spinal cord.</li>
	<li>Gently excise the spinal cord from the vertebral column with blunt tissue forceps and use microscissors to carefully sever the roots to release the cord. Place the excised spinal cord in a Petri dish containing 4 &#176;C sterile rat dissection buffer (described above in 1.1). Rinse the tissue in freshly prepared 4 &#176;C dissecting buffer and using scissors cut the spinal cord transversely into 1 cm segments (Figure 1E).</li>
	<li>For each segment of tissue, use one hand to hold the tissue with fine forceps. With the other hand, use microscissors to carefully remove the overlying meninges, white matter, and most of the grey matter with the aid of a dissecting scope. Alternatively, use fine forceps (Dumont #4) to peel away most of the grey and white matter, leaving only the periventricular region of the spinal cord which includes the ependyma and a small amount of surrounding grey matter (Figure 1F-H).</li>
	<li>Pool the dissected periventricular tissue into a 10 cm sterile Petri dish containing cold rat dissection buffer.</li>
</ol>
3. Harvesting and Dissection of Adult Human Periventricular Spinal Cord Tissue
Note: Human spinal cord tissue is harvested from adult organ transplant donors (donors ranged in age from 2 to 60 years of age) after the other organs have been removed for organ transplantation. The Trillium-Gift of Life Program obtains consent for removal of tissue for research purposes from the patient&#8217;s family and notifies our harvesting team in a timely fashion such that we have been able to harvest the cords within 2 hrs of aortic cross-clamping. Male and female adult donors with negative serology and no infections are accepted.
<ol>
	<li>Harvest tissue in a sterile fashion in the operating room through an anterior approach using the same anterior exposure already dissected for organ removal by the organ transplant harvesting team.</li>
	<li>Expose the anterior spinal column through retraction with large rib spreaders and large paddle retractors of the remaining tissues and organs including the large blood vessels.</li>
	<li>Use a sternal saw mounted with a long blade to cut through the intervertebral discs at the rostral and caudal ends of the desired length of the vertebral column to be resected. Angle the saw about 45&#176; medially to cut a triangular trough from the lateral part of the vertebral bodies stopping just short of the spinal canal.</li>
	<li>Remove en bloc the medial aspects of the vertebral bodies of the desired segments with the aid of curved osteotomes and mallet taking care to avoid cutting through the dura. Then gently lift the dura covered spinal cord with toothed forceps and sharply incise the rostral and caudal ends of the cord. Use long scissors and forceps to bilaterally transect the individual roots.</li>
	<li>Place the excised segment of the spinal cord in 4 &#176;C sterile buffer (1x HBSS + 0.6% glucose + 2% penicillin-streptomycin) in a large test tube for transportation to the tissue culture room.</li>
	 
	<li>Note: Generally, a 3 - 6 cm segment of spinal cord from the upper thoracic and/or mid-to-low thoracic level is removed in the operating room under sterile conditions as soon as possible after cessation of circulation and placed in cold sterile buffer. The time elapsed between cessation of circulation and removal of the spinal cord varies with the time required to harvest the targeted organs for donation and has ranged from 45 min to 2 hrs. If the heart and lungs have also been removed, the dissection can be taken far rostrally to harvest a portion of the lower cervical cord as well.</li>
	<li>Dissect the human spinal cord tissue as soon as possible after harvesting, generally within 3 hrs. Remove the dura and other meninges with forceps. Rinse the spinal cord tissue in freshly made 4 &#176;C dissection buffer and cut transversely into 1 cm segments.</li>
	<li>With the aid of a dissecting scope, excise the overlying meninges, white matter, and most of the grey matter using tissue forceps, fine forceps and microscissors for each tissue segment. Pool the dissected periventricular tissue, which includes the ependyma and a small amount of surrounding grey matter, into a 10 cm sterile Petri dish containing cold human dissection buffer.</li>
</ol>
4. Isolation and Culturing of Adult Rat and Human Spinal Cord NSPCs
<ol>
	<li>Perform the following steps aseptically in a laminar flow hood.
	<ol>
		<li>Mince the dissected rat or human periventricular tissue into 1 mm3 pieces with microscissors.</li>
		<li>Enzymatically dissociate the minced tissue with proteolytic enzymes using the papain dissociation kit as indicated in the table of materials/reagents. Note: Reagent components include 4 vials; Vial 1: Earle&#39;s Balanced Salt Solution (EBSS), Vial 2: Papain containing L-cysteine and EDTA, Vial 3: Deoxyribonuclease I (DNase), Vial 4: Ovomucoid protease inhibitor with bovine serum albumin (BSA). 
		Note: Note: Papain is a sulfhydryl protease that has wide specificity for protein substrates. The papain herein is derived from the latex from the Carica papaya plant and degrades most protein substrates more extensively than pancreatic proteases. During the dissociation process there is some cell damage causing DNA to be released into the dissociation medium which will increase viscosity and make pipetting difficult. Thus, DNase is included in the cell isolation procedure to digest the DNA without damaging the intact cells. Ovomucoids are glycoprotein protease inhibitors used to inhibit the papain activity after the dissociation step.</li>
	</ol>
	</li>
	<li>At first use, reconstitute the albumin ovomucoid inhibitor mixture (vial 4) with 32 ml of EBSS (vial 1), and then store at 4 &#176;C and use for subsequent isolations. Note: This yields a solution at an effective concentration of 10 mg of ovomucoid inhibitor and 10 mg of albumin per ml.</li>
	<li>Add 5 ml of EBSS (vial 1) to a papain vial (vial 2), yielding a solution at 20 units of papain/ml in 1 mM L-cysteine with 0.5 mM EDTA. Check that the papain solution appears clear when completely dissolved, otherwise place the vial in a 37 &#176;C water bath for ten minutes until the papain is completely dissolved to ensure full activity of the enzyme.</li>
	<li>Add 500 &#181;l of EBSS (vial 1) to a DNase vial (vial 3) and mix gently as DNase is sensitive to shear denaturation. Add 250 &#181;l of the DNase solution to the vial containing the papain, resulting in a final concentration of approximately 20 units/ml papain and 0.005% DNase. Save the balance of the DNase vial to use later.</li>
	<li>Place the minced rat or human tissue in the papain solution as prepared in step 4.4 above. For the human tissue isolation, split the minced tissue equally between two papain vials (approximately 0.2 g/vial).</li>
	<li>Incubate at 37 &#176;C with constant agitation on a rocker platform to dissociate the tissue in the activated papain solution. Incubate rat tissue for 45 min to 1 hr, and human tissue for 1 to 2 hrs depending on the amount of minced tissue, about 0.2 &#8211; 0.4 g respectively.</li>
	<li>Triturate the mixture with a 10 ml pipette to dissociate any remaining tissue pieces to yield a cloudy cell suspension. Transfer the cell suspension (do not include any pieces of undissociated tissue) into a sterile 15 ml conical tube and centrifuge at 300 x g for 5 min at RT.</li>
	<li>Resuspend the pelleted cells in medium containing ovomucoid, a papain inhibitor.
	<ol>
		<li>Prepare the ovomucoid solution by mixing 2.7 ml EBSS (vial 1) with 300 &#181;l of the reconstituted albumin-ovomucoid inhibitor solution (vial 4) in a 15 ml conical tube. Add 150 &#181;l of DNase solution (vial 3) saved at step 4.4 above.</li>
		<li>Discard the supernatant from the pelleted cells and immediately resuspend the cell pellet in the diluted DNase albumin-inhibitor mixture.&#160;</li>
	</ol>
	</li>
	<li>Separate intact cells from cell membranes by centrifugation through a single step discontinuous density gradient.
	<ol>
		<li>Prepare the discontinuous density gradient by adding 5 ml of albumin-inhibitor solution (vial 4) to a 15 ml tube, and using a 5 ml pipette, gently and slowly layer the cell suspension (prepared as described above in step 4.8) on top of the albumin-inhibitor solution.</li>
		<li>Centrifuge at 70 x g for 6 min at RT.</li>
		<li>Note: The interface between the two layers of the gradient should be clearly visible, although minimal mixing at this boundary does not affect the result. Membrane fragments remain at the interface and dissociated cells pellet at the bottom of the tube.&#160;</li>
	</ol>
	</li>
	<li>For rat cells, discard the supernatant and resuspend the cell pellet in 1 ml of rat EFH medium (pre-warmed at 37 &#176;C). Count live cell density with a haemocytometer and plate the cells into a T25 culture flask at a density of 10 cells/&#181;l in EFH. The typical yield of cells from rat tissue is about 2 x 106 cells with about 80% viability.If clonal cultures are desired, seed cells at a density of less than 10 cells/&#181;l. Incubate the flasks at 37 &#176;C in 5% CO2 and allow the cultures to grow undisturbed for 1 week to avoid aggregation of spheres.</li>
	<li>For human cells, discard the supernatant and resuspend the cell pellet in 10 ml of human EFH medium (pre-warmed at 37 &#176;C). Filter the cell suspension through a 40 &#181;m nylon cell strainer followed with 30 ml of EFH to remove myelin and cell membrane fragments. Centrifuge at 300 x g for 5 minutes and resuspend the cell pellet in 1 ml of EFH medium.</li>
	<li>Count live cell density with a haemocytometer and plate the human cells into 6-well culture plates at a density of 105 cells/well in a total volume of 5 ml EFH/well. The typical yield of cells from human tissue is about 1 x 106 cells with about 70% viability. Pre-coat wells with an adherent substrate such as Matrigel (see Note below). Dilute at a ratio of 40 &#181;l Matrigel : 1 ml SFM.</li>
	<li>Note: Alternatively, other adherent substrates such as poly-D-lysine/laminin, fibronectin, or collagen may be used.</li>
	<li>Incubate the plates at 37 &#176;C in 5% CO2 and allow the cultures to grow undisturbed for 1 week.</li>
</ol>
5. Passaging of Adult rat and human spinal cord NSPCs
<ol>
	<li>Rat NSPCs will form small neurospheres approximately 70 &#181;m in diameter within 1 week of initial seeding; passage rat NSPCs weekly by collecting the cell suspension, centrifuging at 300 x g for 5 min, and mechanically triturating the cell pellet to dissociate the neurospheres.</li>
	<li>Seed the dissociated cells into T25 flasks containing fresh rat EFH medium. Alternatively, use 50% conditioned rat medium for passaging. The typical yield of rat NSPCs at passaging is about 5 x 106 cells which increases exponentially with passage number.</li>
	<li>For the human cultures, one week after the initial plating, replace half of the culture medium with fresh EFH twice weekly to allow the cells to adhere to the substrate. Generally, 1 - 2 weeks later once the cells have attached to the substrate, replace the entire volume of culture medium twice a week with fresh EFH.</li>
	<li>Subculture cells before reaching confluence between 4 - 8 weeks.
	<ol>
		<li>Subculture cells by detaching cells from the substrate enzymatically (see Table of Materials) with 2 ml of enzyme per well incubated for approximately 10 min at 37 &#176;C. Collect cell suspension, centrifuge at 300 x g for 5 min, and gently resuspend the cell pellet in 1 ml of freshly made EFH medium.</li>
		<li>Count live cells with a haemocytometer and plate the cells into 6-well culture plates (pre-coated as above in step 4.12) at a density of 105 cells/well in a total volume of 5 ml EFH/well. The typical yield of human NSPCs at passaging is about 2 x 106 cells and generally this will double with passage. Generally, maintain cultures with 50% conditioned EFH medium 2 - 3 times per week between passaging.</li>
	</ol>
	</li>
</ol>

<ol><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mammalian+neural+stem+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Mammalian neural stem cells.</a> Science. 287, 1433-1438 (2000).</li>
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The authors have nothing to disclose.

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><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&amp;nbsp;</td><td>Life Technologies</td><td>#10888-022</td><td>Culture medium</td></tr><tr><td>L-glutamine, 200 mM</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td># S-5761</td><td>Prepare 7.5% NaHCO&lt;sub&gt;3&lt;/sub&gt; 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 &amp;mu;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 &amp;mu;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 &amp;mu;g/ml stocks in B27 and aliquot; EFH medium&amp;nbsp;</td></tr><tr><td>Heparin, 10,000 U</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 &amp;ndash; 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&amp;nbsp;</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 &amp;amp; Lomb</td><td>N1430</td><td></td></tr><tr><td>10 mm Petri dishes&amp;nbsp;</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 &amp;mu;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 &amp;mu;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...

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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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Research

JoVE Journal

Developmental Biology

12.3K 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. 

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

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

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 ...

Neuroscience

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 ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Medicine

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 ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

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 expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

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 ...

Rat "Brain Milking" for Neural Stem and Oligodendrocyte Progenitor Cell Isolation

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Author Spotlight: Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells

Source: Tsui, Y, et al. <a href="https://app.jove.com/v/55794">Hypoxic Preconditioning of Marrow-derived Progenitor Cells as a Source for the Generation of Mature Schwann Cells.</a> J. Vis. Exp. (2017).
This video demonstrates a procedure for isolating dorsal root ganglion (DRG) neurons from rat embryos and co-culturing them with Schwann cell precursors to generate mature Schwann cells. The isolated DRGs are processed to isolate neurons and glial cells. The isolated cells are cultured to purify the neurons, which are then co-cultured with Schwann cell-like cells (SCLCs) in a co-culture medium. Interaction with the DRG neurons leads to the maturation of SCLCs into Schwann cells.

Generation of Fate-committed Schwann Cells via Coculture with DRG Neurons
1. Preparation of purified rat DRG neurons

	Autoclave all dissection tools ...

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

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