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>

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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. 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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 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 ...

isolation-neural-stemprogenitor-cells-from-periventricular-region

Research

JoVE Journal

Developmental Biology

12.2K ビュー.  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. 

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

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.