This work was funded (in part) by the Helmholtz Alliance ICEMED - Imaging and Curing Environmental Metabolic Diseases, through the Initiative and Network Fund of the Helmholtz Association, a grant from the Else Kroener-Fresenius Foundation, and a grant from the Deutsche Forschungsgemeinschaft (SFB 655: Cells into tissues).

The work adheres to the Declaration of Helsinski and the ARVO Animal Statement. Animals were used for tissue collection and all relevant methods were followed according to the instructions of the animal facility at the University of Dresden. Animals were handled and housed according to the German Federal guidelines for the use and care of laboratory animals, and the study was approved by the Landesdirektion Dresden. Please consult with your institution&#8217;s veterinary policy (IACUC or other board) regarding appropriate euthanasia methodology.
Specific stock and working concentrations of reagents can be found in the Reagent Tables accompanying this protocol.
1. Culture Dish Preparation
<ol>
	<li>Coat dishes with a sufficient volume of 75 &#956;g/ml poly-L-ornithine (i.e. 2 ml/well of a 6-well plate) overnight in the cell culture incubator 2 days prior to the scheduled dissection date.</li>
	<li>Aspirate the poly-L-ornithine the following day and wash each well 2x&#160;with 3 ml of PBS for 5 min.</li>
	<li>Remove the final wash, then add fibronectin (diluted 1:250, 4 &#956;g/ml in PBS) to the plate and place the dishes in the incubator overnight.</li>
	<li>Aspirate the fibronectin on&#160;the day of dissection and wash the dishes 2x with PBS. Return the plates still containing the final PBS wash to the incubator&#160;until the dissociated tissue is ready for culturing. At that time, remove the PBS prior to plating the tissue.</li>
	<li>Store plates coated with poly-L-ornithinein the incubator for up to 3 weeks. Plates coated with fibronectin can be stored for up to 1 week. Fibronectin coating can be done in as little as 2 hr if urgent. Fibronectin is susceptible to denaturation; do not agitate the solution or let dishes dry.</li>
</ol>
2. Dissection/Plating
<ol>
	<li>This protocol applies to the use of 3 adult rats (3-6 months of age).</li>
	<li>Chill the brain sectioning block on ice prior to euthanizing the rat.</li>
	<li>Place a 15&#160;ml Falcon tube that contains 5 ml of N2 medium with added bFGF on ice.</li>
	<li>Euthanize the rats according to your institution&#8217;s veterinary policy.</li>
	<li>Remove the brain from the skull by making an incision down the midline of the head. Use forceps to peel away the bone allowing for the brain to be carefully extracted.</li>
	<li>Place the brain into a 10 cm Petri dish containing ice cold PBS. This will remove any residual hair and blood. (Figure 1A)</li>
	<li>Place the brain into the chilled sectioning block. (Figure 1B)</li>
	<li>Insert one new clean razor blade into the block as pictured in Figure 1C.</li>
	<li>Insert a second clean razor blade 3 mm caudal to the first as illustrated in Figure 1D.</li>
	<li>Carefully lift the blades from the block, taking with them the section of interest.</li>
	<li>Float the section off the blade by immersing it in PBS. (Figure 1E)</li>
	<li>Harvest tissue from the area of interest (in this example we used the anterior commissure, (anterior) ACA, Figure 1F) using #5 forceps and collect into a 15 ml conical tube containing 1 ml N2 medium containing bFGF.</li>
	<li>Move the tube into a laminar flow cell culture hood, continuing the remainder of the protocol under sterile conditions.</li>
	<li>Mechanically dissociate the tissue using a 1 ml pipette tip attached to a P1000 pipettor. Pipette up and down several times (approximately 20x at a rate of 1 pipetting cycle per second) while pressing the opening of the pipette tip against the bottom of the conical tube&#160;(Figures&#160;1G 1-3). Stop trituration when the solution becomes homogeneous.</li>
	<li>Allow the solution to sit for 2 min to allow any larger nondissociated tissue to settle to the bottom. (Figure 1G 4)</li>
	<li>Collect the homogeneous supernatant and place into a conical tube containing 8.5 ml of N2 media plus 20 ng/ml bFGF, 500 ng/ml Dll4, 500 ng/ml Ang2, and 200 nMJAK Inhibitor.</li>
	<li>Plate them into 3 wells of a 6-well plate using 3 ml of diluted cell solution/well.</li>
	<li>Place the plate into a humidified cell incubator at 37 &#176;C, 5% CO2, 5% O2.</li>
</ol>
3. Daily Care
<ol>
	<li>Replace the medium on the culture dishes with N2 Medium containing bFGF, Ang2, Dll4, and JAK inhibitor 24 hr after plating.</li>
	<li>Add a bolus of bFGF, Dll4, Ang2, and JAK inhibitor on the next day.</li>
	<li>Continue this alternating process of media changes and factor additions for a total of 10-14 days.</li>
</ol>
4. Induction of Differentiation
<ol>
	<li>Withdraw bFGF, Dll4, Ang2, and JAK inhibitor containing medium and replace it with N2 medium that does not contain any additional factors.</li>
	<li>Change the medium every second day.</li>
	<li>Fix the cells after 10 days and perform immunocytochemistry.</li>
</ol>
5. Immunocytochemical Detection
<ol>
	<li>Aspirate the medium and fix cell culture plates with 2 ml of 4% paraformaldehyde for 20 min.</li>
	<li>Wash 2x with 3 ml of PBS for 5 min each.</li>
	<li>Aspirate PBS, then permeabilize and block the cells by adding 2 ml of PBS containing 5% normal donkey serum (NDS) and 0.1% Triton X-100 for 20 min.</li>
	<li>Prepare the primary antibody mix in PBS containing 5% NDS. Anti-nestin antibody can be used for identifying SCs, while TuJ1 (class III &#946;-tubulin), GFAP, and CNPase antibodies can be used together for a triple staining of differentiation markers.</li>
	<li>Aspirate the blocking solution and add 1.5 ml of the primary antibody mixture to each well. Incubate at room temperature for 90 min.</li>
	<li>Remove the primary antibodies and wash 2x with 3 ml of PBS, and 1x with 3 ml of PBS containing 5% NDS&#160;for 5 min each.</li>
	<li>Prepare the secondary antibodies in PBS containing 5% NDS.</li>
	<li>Aspirate the final wash and add 1.5 ml of secondary antibody solution to each well. Incubate in the dark for 40 min at room temperature.</li>
	<li>Remove the secondary antibody solution and add 2 ml of a freshly prepared DAPI stain (500 ng/ml in PBS). Incubate for 3 min.</li>
	<li>Aspirate the DAPI solution then wash 3x with 3 ml of PBS for 5 min&#160;each. The cells can now be visualized with a fluorescence microscope.</li>
</ol>

<ol>
	<li>Cajal, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comparative Study of Sensory Areas of the Human Cortex. , (1899).</li><li>Altman, J., Das, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiographic+and+histological+evidence+of+postnatal+hippocampal+neurogenesis+in+rats.">Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats.</a> J. Comp. Neurol. 124, 319-335 (1965).</li><li>Kaplan, M. S., Hinds, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+adult+rat:+electron+microscopic+analysis+of+light+radioautographs.">Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs.</a> Science. 197, 1092-1094 (1977).</li><li>Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+replacement+in+adulthood.">Neuronal replacement in adulthood.</a> Ann. N.Y. Acad. Sci. 457, 143-161 (1985).</li><li>Gould, E., Reeves, A. J., Graziano, M. S., Gross, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+neocortex+of+adult+primates.">Neurogenesis in the neocortex of adult primates.</a> Science. 286, 548-552 (1999).</li><li>Kornack, D. R., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuation+of+neurogenesis+in+the+hippocampus+of+the+adult+macaque+monkey.">Continuation of neurogenesis in the hippocampus of the adult macaque monkey.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 5768-5773 (1999).</li><li>Alvarez-Buylla, A., Kohwi, M., Nguyen, T. M., Merkle, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heterogeneity+of+adult+neural+stem+cells+and+the+emerging+complexity+of+their+niche.">The heterogeneity of adult neural stem cells and the emerging complexity of their niche.</a> Cold Spring Harb. Symp. Quant. Biol. 73, 357-365 (2008).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Luskin, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+proliferation+and+migration+of+postnatally+generated+neurons+derived+from+the+forebrain+subventricular+zone.">Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone.</a> Neuron. 11, 173-189 (1993).</li><li>Lendahl, U., Zimmerman, L. B., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+stem+cells+express+a+new+class+of+intermediate+filament+protein.">CNS stem cells express a new class of intermediate filament protein.</a> Cell. 60, 585-595 (1990).</li><li>Androutsellis-Theotokis, 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=Targeting+neural+precursors+in+the+adult+brain+rescues+injured+dopamine+neurons.">Targeting neural precursors in the adult brain rescues injured dopamine neurons.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 13570-13575 (2009).</li><li>Jin, K., 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=Neurogenesis+in+dentate+subgranular+zone+and+rostral+subventricular+zone+after+focal+cerebral+ischemia+in+the+rat.">Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 4710-4715 (2001).</li><li>Zhang, R. L., Zhang, Z. G., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+stroke+and+neurogenesis+in+the+subventricular+zone.">Ischemic stroke and neurogenesis in the subventricular zone.</a> Neuropharmacology. 55, 345-352 (2008).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+are+MSCs+therapeutic?+New+data:+new+insight.">Why are MSCs therapeutic? New data: new insight.</a> J. Pathol. 217, 318-324 (2009).</li><li>Goldman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+and+progenitor+cell-based+therapy+of+the+human+central+nervous+system.">Stem and progenitor cell-based therapy of the human central nervous system.</a> Nat. Biotechnol. 23, 862-871 (2005).</li><li>Sun, N., Longaker, M. T., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPS+cell-based+therapy:+considerations+before+clinical+applications.">Human iPS cell-based therapy: considerations before clinical applications.</a> Cell Cycle. 9, 880-885 (2010).</li><li>Amariglio, N., 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=Donor-derived+brain+tumor+following+neural+stem+cell+transplantation+in+an+ataxia+telangiectasia+patient.">Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient.</a> PLoS Med. 6, e1000029 (2009).</li><li>Lindvall, O., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapeutics+in+Parkinson's+disease.">Cell therapeutics in Parkinson's disease.</a> Neurotherapeutics. 8, 539-548 (2011).</li><li>Kittappa, R., Bornstein, S. R., Androutsellis-Theotokis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+eNSCs+in+Neurodegenerative+Disease.">The Role of eNSCs in Neurodegenerative Disease.</a> Mo.l Neurobiol. , (2012).</li><li>Cattaneo, E., McKay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+and+manipulating+neuronal+stem+cells.">Identifying and manipulating neuronal stem cells.</a> Trends Neurosci. 14, 338-340 (1991).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol. Neurobiol. 34, 153-161 (2006).</li><li>Duarte, 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=Dosage-sensitive+requirement+for+mouse+Dll4+in+artery+development.">Dosage-sensitive requirement for mouse Dll4 in artery development.</a> Genes Dev. 18, 2474-2478 (2004).</li><li>Maisonpierre, P. 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=Angiopoietin-2,+a+natural+antagonist+for+Tie2+that+disrupts+in+vivo+angiogenesis.">Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis.</a> Science. 277, 55-60 (1997).</li><li>Androutsellis-Theotokis, 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=Notch+signalling+regulates+stem+cell+numbers+in+vitro+and+in+vivo.">Notch signalling regulates stem cell numbers in vitro and in vivo.</a> Nature. 442, 823-826 (2006).</li><li>Alderson, R. F., Alterman, A. L., Barde, Y. A., Lindsay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+increases+survival+and+differentiated+functions+of+rat+septal+cholinergic+neurons+in+culture.">Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture.</a> Neuron. 5, 297-306 (1990).</li><li>Johe, K. K., Hazel, T. G., Muller, T., Dugich-Djordjevic, M. M., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+factors+direct+the+differentiation+of+stem+cells+from+the+fetal+and+adult+central+nervous+system.">Single factors direct the differentiation of stem cells from the fetal and adult central nervous system.</a> Genes Dev. 10, 3129-3140 (1996).</li><li>Ourednik, J., Ourednik, V., Lynch, W. P., Schachner, M., Snyder, E. Y. <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+display+an+inherent+mechanism+for+rescuing+dysfunctional+neurons.">Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons.</a> Nat. Biotechnol. 20, 1103-1110 (2002).</li><li>Ryu, J. K., Cho, T., Wang, Y. T., McLarnon, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cells+attenuate+inflammatory+reactivity+and+neuronal+loss+in+an+animal+model+of+inflamed+AD+brain.">Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain.</a> J. Neuroinflammation. 6, 39 (2009).</li><li>Androutsellis-Theotokis, 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=Angiogenic+factors+stimulate+growth+of+adult+neural+stem+cells.">Angiogenic factors stimulate growth of adult neural stem cells.</a> PLoS One. 5, e9414 (2010).</li><li>Park, D. 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=Hes3+regulates+cell+number+in+cultures+from+glioblastoma+multiforme+with+stem+cell+characteristics.">Hes3 regulates cell number in cultures from glioblastoma multiforme with stem cell characteristics.</a> Sci. Rep. 3, 1095-1010 (2013).</li><li>Poser, S. W., Shostak, 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=Ch.+5.">Ch. 5.</a> Cancer Stem Cells - The Cutting. , 89-110 (2011).</li><li>Androutsellis-Theotokis, A., Rueger, M. A., Mkhikian, H., Korb, E., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+controlling+neural+stem+cells+slow+progressive+brain+disease.">Signaling pathways controlling neural stem cells slow progressive brain disease.</a> Cold Spring Harb. Symp. Quant. Biol. 73, 403-410 (2008).</li></ol>

We have nothing to disclose.

Many of the steps critical to successful isolation, expansion, and differentiation of neural stem cells from the adult brain are shared in common with standard tissue culture techniques. The objective to bear in mind is that NSCs isolated from the adult brain should maintain as many of their in vivo&#160;characteristics as possible (Figure 2J). Often a balance between necessary caution and appropriate speed needs to be struck. Care with the isolation of the brain from the skull will make the ide...

The central dogma of neurobiology, laid down by the fundamental observations of the brain cytoarchitecture made by Ram&#243;n Y. Cajal over a century ago, held that neurogenesis was unlikely after adolescence given the complexity of the neural networks found in the CNS1. Despite the work of Altman in the 1960s, and later Kaplan, demonstrating that 3H-thymidine could be found in mature neurons indicating that in fact neurons were being generated in distinct areas of the adult brain, the dogma continued to hold2,3. Evidence continued to mount with Nottebohm&#8217;s research describing the seasonal changes in the number of neurons present in songbird brains4. It wasn&#8217;t until 1999, when Gould et al. published work on the generation of neurons in the hippocampus increases with the performance of associative learning tasks in rat, as well as the observations of Kornack and Rakic demonstrating continued neurogenesis in the adult macaque that the concept of a less rigid, more plastic brain, was recognized5,6.
The search for the cellular source for these de novo generated neurons lead to the discovery of a discrete population of stem cells (SCs) that reside in areas of the brain referred to as niches7. The subventricular zone and sub granular zone of the hippocampus are considered to be the two principal neurogenic regions8,9. Cells isolated from these locations display the classic characteristic of embryonic or fetal derived SCs, self-renewal and differentiation potential. In the case of neural stem cells (NSCs), they can be differentiated into neurons, astrocytes, and oligodendrocytes. In addition, these SCs &#160;stain positive for fetal NSC markers such as the intermediate filament protein nestin10. More recent work highlights that SCs might not be limited to these two areas, and are in fact localized throughout the brain as a largely quiescent population of cells tightly associated with the vasculature11.
The observations that SCs are mobilized in response to injury suggests the possibility of being able to utilize these cells for regenerative purposes to aid in the recovery from neurodegenerative disorder and stroke12,13. This is not unlike the role that mesenchymal stem cells (MSCs) &#160;play in the healing of connective tissues, which are found as perivascular cells that have the potential to become osteoblasts, chondrocytes, and adipose cells14. However, NSCs cannot be harvested in the same manner as MSCs from bone marrow by routine aspiration and density gradient centrifugation techniques and subsequently utilized in autologous cell based therapies. As a consequence, other sources of cells, such as the use of fetal NSCs or neuronal precursors derived from embryonic stem cells have been extensively explored in animal disease and injury models with varying degrees of success15. Induced pluripotent stem cell technologies utilizing somatic cell sources offer another potential avenue for producing therapeutically useful cell-based therapies for a wide range of applications, overcoming the limited availability and ethical concerns regarding the use of embryonic cells and fetal tissues16. However, clinical translation of these findings has proven to be a difficult task, as demonstrated in the struggles of treating various neurological conditions with SC-based therapeutic approaches17,18, as well as a tortuous path to regulatory clearance. As an alternative approach, introduction of specific pharmacological treatments can modulate NSC numbers and facilitate recovery in models of Parkinson&#8217;s disease and stroke19. Whatever the strategy might be, understanding how to effectively manipulate these cells requires an accessible in vitro system.
Cultures of NSCs can be performed either as aggregate cultures, also known as neurospheres, or as a monolayer8,20. Both techniques have been widely used, allowing for the establishment of defined culture conditions, i.e. use of Epidermal Growth Factor (EGF) or basic Fibroblast Growth Factor (bFGF) as a mitogen source, that provide for the expansion of multipotent precursors. While neurosphere cultures may be better suited for studying clonal propagation capability of an isolated cell type, the system has been shown to produce a mixed population of cells during expansion21. In addition, the closed structure of neurospheres makes pharmacological manipulation of the cells impractical, and the interpretation of the influence these factors may have could be confounded due to the microenvironment established within the neurosphere itself. Monolayer cultures, on the other hand, can be employed in high throughput screens of small molecule libraries, providing a powerful tool to explore the signal transduction mechanisms that regulate SC growth and differentiation and opens the opportunity to discover novel compounds that specifically target this cell population.
As a consequence, the ability to reproducibly generate cultures of adult NSCs from different regions of interest in the brain can be used in a broad spectrum of research applications, ranging from developmental studies of the central nervous system (CNS) to exploring novel regenerative medicine approaches. The protocol presented here demonstrates how to dissect and assess the differentiation potential of CNS SCs isolated from the adult rodent brain.

<table><tbody><tr><td>6-well tissue culture dishes</td><td>BD Biosciences</td><td>353934</td><td></td></tr><tr><td>Poly-L-ornithine</td><td>Sigma-Aldrich</td><td>P-3655</td><td> &lt;p&gt;5 mg/ml stock solution prepared in double distilled water (Stable for several months at -20 &amp;deg;C).&lt;/p&gt; &lt;p&gt;Working concentration 0.5 mg/ml in water (Stable for 1 month at 4 &amp;deg;C).&lt;/p&gt; </td></tr><tr><td>Fibronectin</td><td>R&amp;amp;D Systems</td><td>1030-Fn</td><td>Do not agitate stock solution</td></tr><tr><td>Filtration Apparatus</td><td>Corning Life Sciences</td><td>430769</td><td></td></tr><tr><td>DMEM/F-12</td><td>Mediatech</td><td>10-090-CV</td><td>See note below for complete N2 media preparation</td></tr><tr><td>Apo-transferrin</td><td>Sigma-Aldrich</td><td>T-2036</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I-0516</td><td></td></tr><tr><td>Putrescine</td><td>Sigma-Aldrich</td><td>P-5780</td><td>1 M stock solution in ddH&lt;sub&gt;2&lt;/sub&gt;O (Stable at 20 &amp;deg;C for 6 months)</td></tr><tr><td>Sodium Selenite</td><td>Sigma-Aldrich</td><td>S-5261</td><td>500 &amp;micro;M stock solution in ddH&lt;sub&gt;2&lt;/sub&gt;O (Stable at -20 &amp;deg;C for 6 months)</td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P-8783</td><td>100 &amp;micro;M stock solution in ethanol (Stable at -20&amp;deg;C for 6 months)</td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Mediatech</td><td>21-040-CV</td><td></td></tr><tr><td>Basic Fibroblast Growth Factor (bFGF)</td><td>R&amp;amp;D Systems</td><td>233-FB</td><td>Working concentration 20 ng/ml&amp;nbsp;</td></tr><tr><td>Delta4 (Dll4)</td><td>R&amp;amp;D Systems</td><td>1389-D4</td><td>Working concentration&amp;nbsp; 500 ng/ml</td></tr><tr><td>Angiopoetin 2 (Ang2)</td><td>R&amp;amp;D Systems</td><td>623-AN</td><td>Working concentration&amp;nbsp; 500 ng/ml</td></tr><tr><td>JAK Inhibitor</td><td>Calbiochem</td><td>420099</td><td>Working concentration 200 nM&amp;nbsp;</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A-2058</td><td></td></tr><tr><td>15- and 50-ml Conical tubes</td><td>Corning Life Sciences</td><td>430053, 430829</td><td></td></tr><tr><td>Other necessary items include: General dissection instruments, including razor blade and forceps, Adult rat (3-6 months old; Sprague-Dawley or Long Evans), CO&lt;sub&gt;2&lt;/sub&gt; intoxication chamber, Laminar flow hood for cell culture and incubator Incubator (humidified, 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;, 5% O&lt;sub&gt;2&lt;/sub&gt;). Note: For complete N2 media preparation, to one bottle of DMEM/F-12 (500 ml) add 0.05 g of apotransferin, 0.0125 g of insulin (freshly predissolved in 1 ml of 10 mM NaOH), 50 &amp;mu;l of putrescine, 30 &amp;mu;l of sodium selenite, 100 &amp;mu;l of progesterone stocks, and 5 ml of penicillin/streptomycin solution.&amp;nbsp; Adjust pH to 7.2, if needed. Filter-sterilize and store at 4 &amp;deg;C for up to 3 weeks and protect from light.</td><td></td><td></td><td></td></tr><tr><td colspan="4">Immunofluorescence Reagents Table</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15719</td><td></td></tr><tr><td>Normal Donkey Serum (NDS)</td><td>Sigma-Aldrich</td><td>D-9663</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T-8787</td><td></td></tr><tr><td>4,6-Diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich</td><td>D-8417</td><td>5 mg/ml stock solution in methanol</td></tr><tr><td colspan="4">Primary Antibody Table</td></tr><tr><td>Nestin</td><td>Chemicon</td><td>MAB353</td><td> &lt;p&gt;Dilution Factor: 1:400&lt;/p&gt; &lt;p&gt;Species: Mouse IgG1&lt;/p&gt; </td></tr><tr><td>Hes3</td><td>Santa Cruz</td><td>sc-25393</td><td> &lt;p&gt;Dilution Factor: 1:100&lt;/p&gt; &lt;p&gt;Species: Rabbit IgG&lt;/p&gt; </td></tr><tr><td>Sox2</td><td>R&amp;amp;D Systems</td><td>MAB2018</td><td> &lt;p&gt;Dilution Factor: 1:100&lt;/p&gt; &lt;p&gt;Species: Mouse IgG2a&lt;/p&gt; </td></tr><tr><td>CNPase</td><td>Chemicon</td><td>MAB326</td><td> &lt;p&gt;Dilution Factor: 1:200&lt;/p&gt; &lt;p&gt;Species: Mouse IgG1&lt;/p&gt; </td></tr><tr><td>&amp;beta;-tubulin III (TUJ1)</td><td>R&amp;amp;D Systems</td><td>MAB1195</td><td> &lt;p&gt;Dilution Factor: 1:500&lt;/p&gt; &lt;p&gt;Species: Mouse IgG2a&lt;/p&gt; </td></tr><tr><td>Glial Fibrillary Acidic Protein (GFAP)</td><td>Dako North America</td><td>Z0334</td><td> &lt;p&gt;Dilution Factor 1:500&lt;/p&gt; &lt;p&gt;Species: Rabbit&lt;/p&gt; </td></tr><tr><td colspan="4">Secondary Antibody Table</td></tr><tr><td>Alexa&amp;nbsp;&amp;nbsp; 568</td><td>Invitrogen</td><td>A-21124</td><td> &lt;p&gt;Dilution Factor: 1:200&lt;/p&gt; &lt;p&gt;Species: Goat anti Mouse IgG1&lt;/p&gt; </td></tr><tr><td>Alexa 488</td><td>Invitrogen</td><td>A-21131</td><td> &lt;p&gt;Dilution Factor: 1:200&lt;/p&gt; &lt;p&gt;Species: Goat anti Mouse IgG2a&lt;/p&gt; </td></tr><tr><td>Cy5</td><td>Jackson ImmunoResearch</td><td>59883</td><td> &lt;p&gt;Dilution Factor: 1:200&lt;/p&gt; &lt;p&gt;Species: Goat anti Rabbit&lt;/p&gt; </td></tr></tbody></table>

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.

Identifying the region of interest from which to harvest neural stem cells is the critical first step and will define the amount of time it takes to get a confluent plate of cells. For example, the SVZ is a classic neurogenic area and therefore has a higher relative proportion of neural stem cells. However, the technique presented here can be used with other regions not often recognized as having a robust neurogenic potential during adulthood. For the purposes of this protocol, we used the anterior commissure (anterior p...

Watch this Scientific Journal Video about Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain at JoVE.com

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

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 ...

growing-neural-stem-cells-from-conventional-nonconventional-regions

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

12.2K Views.  University of Dresden. 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. 

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

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.

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 ...

Developmental Biology

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

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

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

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

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 ...

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

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

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

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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 ...

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 ...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...