This work was supported by IF/01227/2015 and UID/BIM/50005/2019, projeto financiado pela Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT)/ Minist&#233;rio da Ci&#234;ncia, Tecnologia e Ensino Superior (MCTES) atrav&#233;s de Fundos do Or&#231;amento de Estado. R.S. (SFRH/BD/128280/2017, F.F.R. (IMM/CT/35-2018), D.M.L. (PD/BD/141784/2018), and R.S.R. (SFRH/BD/129710/2017) received a fellowship from FCT. The authors would like to thank the members of the bioimaging facility at Instituto de Medicina Molecular Jo&#227;o Lobo Antunes for microscopy assistance.

<ol>
	<li>Reynolds, B., 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 (5052), 1707-1710 (1992).</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> Journal of Visualized Experiments. (45), e2393 (2010).</li><li>Vescovi, A. L., Reynolds, B. A., Fraser, D. D., 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=bFGF+regulates+the+proliferative+fate+of+unipotent+(neuronal)+and+bipotent+(neuronal/astroglial)+EGF-generated+CNS+progenitor+cells.">bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells.</a> Neuron. 11 (5), 951-966 (1993).</li><li>Rietze, R. L., 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=Neural+Stem+Cell+Isolation+and+Characterization.">Neural Stem Cell Isolation and Characterization.</a> Methods in Enzymology. 419, 3-23 (2006).</li><li>Walker, T. L., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+Mouse,+Two+Cultures:+Isolation+and+Culture+of+Adult+Neural+Stem+Cells+from+the+Two+Neurogenic+Zones+of+Individual+Mice.">One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice.</a> Journal of Visualized Experiments. (84), e51225 (2014).</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> Molecular Neurobiology. 34 (3), 153-162 (2006).</li><li>Xapelli, 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=Activation+of+Type+1+Cannabinoid+Receptor+(CB1R)+Promotes+Neurogenesis+in+Murine+Subventricular+Zone+Cell+Cultures.">Activation of Type 1 Cannabinoid Receptor (CB1R) Promotes Neurogenesis in Murine Subventricular Zone Cell Cultures.</a> PLoS ONE. 8 (5), e63529 (2013).</li><li>Riccardi, C., Nicoletti, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+apoptosis+by+propidium+iodide+staining+and+flow+cytometry.">Analysis of apoptosis by propidium iodide staining and flow cytometry.</a> Nature Protocols. 1 (3), 1458-1461 (2006).</li><li>Nowakowski, R. S., Lewin, S. B., Miller, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+immunohistochemical+determination+of+the+lengths+of+the+cell+cycle+and+the+DNA-synthetic+phase+for+an+anatomically+defined+population.">Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population.</a> Journal of Neurocytology. 18 (3), 311-318 (1989).</li><li>Weinberg, D., Adams, C. F., Chari, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deploying+clinical+grade+magnetic+nanoparticles+with+magnetic+fields+to+magnetolabel+neural+stem+cells+in+adherent+versus+suspension+cultures.">Deploying clinical grade magnetic nanoparticles with magnetic fields to magnetolabel neural stem cells in adherent versus suspension cultures.</a> RSC Advances. 5 (54), 43353-43360 (2015).</li><li>Azari, H., Sharififar, S., Rahman, M., Ansari, 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=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> Journal of Visualized Experiments. (47), e2457 (2011).</li><li>Azari, H., Louis, S. A., Sharififar, S., Vedam-Mai, V., 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=Neural-Colony+Forming+Cell+Assay:+An+Assay+To+Discriminate+Bona+Fide+Neural+Stem+Cells+from+Neural+Progenitor+Cells.">Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells.</a> Journal of Visualized Experiments. (49), e2639 (2011).</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> Nature Methods. 2 (5), 333-336 (2005).</li><li>Pastrana, E., Cheng, L. C., Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+prospective+purification+of+adult+subventricular+zone+neural+stem+cells+and+their+progeny.">Simultaneous prospective purification of adult subventricular zone neural stem cells and their progeny.</a> Proceedings of the National Academy of Sciences. 106 (15), 6387-6392 (2009).</li><li>Conti, L., Cattaneo, E. <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+cell+systems:+physiological+players+or+in+vitro+entities?.">Neural stem cell systems: physiological players or in vitro entities?.</a> Nature Reviews Neuroscience. 11 (3), 176-187 (2010).</li><li>Ghate, P. S., Sidhar, H., Carlson, G. A., Giri, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+cellular+model+of+Alzheimer's+disease+utilizing+neurosphere+cultures+derived+from+B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J+embryonic+mouse+brain.">Development of a novel cellular model of Alzheimer's disease utilizing neurosphere cultures derived from B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J embryonic mouse brain.</a> SpringerPlus. 3 (1), 161 (2014).</li><li>Pluchino, 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=Injection+of+adult+neurospheres+induces+recovery+in+a+chronic+model+of+multiple+sclerosis.">Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis.</a> Nature. 422 (6933), 688-694 (2003).</li><li>Ogawa, Y., Kaizu, K., Yanagi, Y., Takada, S., Sakuraba, H., Oishi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+differentiation+of+Sandhoff+disease+model+mouse-derived+multipotent+stem+cells+toward+a+neural+lineage.">Abnormal differentiation of Sandhoff disease model mouse-derived multipotent stem cells toward a neural lineage.</a> PLoS ONE. 12 (6), e0178978 (2017).</li><li>Khacho, 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=Mitochondrial+Dynamics+Impacts+Stem+Cell+Identity+and+Fate+Decisions+by+Regulating+a+Nuclear+Transcriptional+Program.">Mitochondrial Dynamics Impacts Stem Cell Identity and Fate Decisions by Regulating a Nuclear Transcriptional Program.</a> Cell Stem Cell. 19 (2), 232-247 (2016).</li><li>Soares, R., 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=Tauroursodeoxycholic+Acid+Enhances+Mitochondrial+Biogenesis,+Neural+Stem+Cell+Pool,+and+Early+Neurogenesis+in+Adult+Rats.">Tauroursodeoxycholic Acid Enhances Mitochondrial Biogenesis, Neural Stem Cell Pool, and Early Neurogenesis in Adult Rats.</a> Molecular Neurobiology. 55 (5), 3725-3738 (2017).</li><li>Rodrigues, R. S., Ribeiro, F. F., Ferreira, F., Vaz, S. H., Sebasti&#227;o, A. M., Xapelli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+Cannabinoid+Type+1+and+Type+2+Receptors+in+the+Modulation+of+Subventricular+Zone+and+Dentate+Gyrus+Neurogenesis.">Interaction between Cannabinoid Type 1 and Type 2 Receptors in the Modulation of Subventricular Zone and Dentate Gyrus Neurogenesis.</a> Frontiers in Pharmacology. 8, 516 (2017).</li><li>Xapelli, 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=Modulation+of+subventricular+zone+oligodendrogenesis:+a+role+for+hemopressin?.">Modulation of subventricular zone oligodendrogenesis: a role for hemopressin?.</a> Frontiers in Cellular Neuroscience. 8, 59 (2014).</li><li>Kim, H. J., 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=Dynamin-related+protein+1+controls+the+migration+and+neuronal+differentiation+of+subventricular+zone-derived+neural+progenitor+cells.">Dynamin-related protein 1 controls the migration and neuronal differentiation of subventricular zone-derived neural progenitor cells.</a> Scientific Reports. 5 (1), 15962 (2015).</li></ol>

The authors have nothing to disclose.

In vitro systems of NSPCs allow a better understanding of the cellular and molecular mechanisms, which can be further validated in vivo. The NSA is a very powerful method to mimic physiological conditions due to their three-dimensional structure. Moreover, this culture system is also technically easier to culture10, compared with other in vitro systems such as the monolayer culture system. Indeed, with the NSA, it is easy to control the exposed extrinsic cues during cell development, either during the expansion or the differentiation phase, by adding precise and variable amounts of factors of interest to the media as well as by culturing neurospheres with other cell types6. Furthermore, compared with monolayer cultures, in the NSA, it is possible to obtain a higher cell density from a small amount of tissue or with a small number of cells, allowing parallel studies to be performed, thus reducing the number of animals1.
The NSA is the most common method to isolate and expand NSCs11,12,13, can be used to estimate the number of precursor cells present in a given tissue sample5 and the precursor cell frequency between different conditions. However, both neurospheres and monolayer cultures do not account for quiescence NSCs14. Moreover, the NSA has some limitations11,12,13 and the resulting neurosphere frequency depends on many factors including the medium components, the dissection procedure, the dissociation process11,12,13, and neurosphere aggregation5. Indeed, in a high-density culture, neurospheres tend to aggregate. Consequently, caution must be taken when estimating the number of precursor cells in a sample. To overcome the above limitations, isolated NSPCs can also be expanded and passaged in a monolayer5,15. Importantly, using NSA to compare precursor cell frequency between different conditions is very useful and accurate because all these limitations are implicit and similar among all conditions performed in the same experiment.
There are critical steps in the neurosphere culture that need attention. In the brain harvesting step, complete removal of the meninges and good isolation of the neurogenic niches are essential to maximize the purity and yield of NSPCs. During tissue dissociation, due to the proteolytic activity of trypsin, excessive use of trypsin or longer incubation times can lead to cell lysis. Furthermore, the day of the passage is critical to obtain a healthy population of neurospheres. Passaging neurospheres with a diameter higher than 200 &#181;m greatly affects the viability, proliferative and differentiative capacity of NSPCs. Importantly, longer cycles of passages, more than 10 can increase genetic instability6. Furthermore, coating with PDL and PLD/laminin for SVZ and DG cells, respectively, is essential to ensure good cell migration out of the neurospheres without compromising the differentiation process. In terms of the immunocytochemistry analysis, longer incubation times with PFA can compromise staining by masking the antigens and increasing the background.
The NSA is&#160;a powerful tool for providing a consistent and an unlimited source of NSPCs for in vitro studies of neural development and differentiation as well as for therapeutic purposes16,17. Indeed, this assay can be applied to genetic and behavioral models to further understand the molecular and cellular mechanisms involved in NSPC proliferation and differentiation18,19. This assay is also useful to test different drugs and compounds20,21,22 as well as to perform genetic manipulations19,23 to modulate NSC properties. In addition to immunocytochemistry, reverse transcription polymerase chain reaction and Western blot analysis can be performed to access RNA and protein expression, while electrophysiological studies and calcium imaging can be used to evaluate the function of the new-born neurons21.

The neurosphere assay (NSA) was firstly described in 19921,2 and still remains a unique and powerful tool in neural stem cell (NSC) research. The isolation of NSCs from the main neurogenic regions has challenging issues because the requirements to maintain these cells in physiological conditions remain poorly understood. In the NSA, cells are cultured in a chemically defined serum-free medium with the presence of growth factors including the epidermal growth factor (EGF) and the basic fibroblast growth factor (bFGF)1,2,3. Neural precursor cells (stem and progenitors) are selected by using these mitogens since these cells are EGF and FGF-responsive entering a period of active proliferation while other cells, namely differentiated cells, die4. Neural precursor cells grow as neurospheres, which can be then passaged to further expand the pool of these cells5. Importantly, since these neural stem progenitor cells (NSPCs) are multipotent they are able to differentiate into the three major cell types of the central nervous system (CNS): neurons, oligodendrocytes and astrocytes5.
The NSA provides a renewable source of undifferentiated CNS precursors, which can be used to study several processes including NSC proliferation and self-renewal, and neuronal and glial differentiation, in both physiologic and disease context. Moreover, in vitro studies can be used to evaluate the degree of intrinsic specification present in neural precursors during development, as well as to study the full potential of the cells, by removing extrinsic cues associated with their normal environment6. The neurosphere model is valuable to evaluate putative regulators since by maintaining the cells in a medium devoid of serum, the environmental cues are only provided by the surrounding cells6. Moreover, in the NSA, NSPCs are easily expanded in culture, the density of cells per area is high and the heterogeneous composition of the neurospheres has some similarity to in vivo niches6. These well-established advantages are the reason why this methodology has been widely used by many researchers.
The following protocol describes in detail all the processes from the isolation of postnatal NSPC population from the two main neurogenic regions, the subventricular zone (SVZ) and the hippocampal dentate gyrus (DG), to the expansion of those cells as neurospheres, as well as to the differentiation into neurons, astrocytes and oligodendrocytes. Lastly, different assays are also described to access stemness and multipotency properties of SVZ- and DG-derived NSPCs.

<table><tbody><tr><td>0.05% Trypsin-EDTA (1X)</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>0.4% Trypan Blue solution</td><td>Sigma-Aldrich</td><td>T8154-20ML</td><td></td></tr><tr><td>12mm Glass coverslips</td><td>VWR</td><td>631-1577</td><td></td></tr><tr><td>15mL Centrifuge Tube</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>5-bromo-2&#039;-deoxyuridine</td><td>Sigma-Aldrich</td><td>B9285-1G</td><td></td></tr><tr><td>50 mL Centrifuge Tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>70% Ethanol</td><td>Manuel Vieira &amp;amp; C&amp;ordf; (Irm&amp;atilde;o) Sucrs, Lda</td><td>UN1170</td><td></td></tr><tr><td>Adhesion slides, Menzel Gl&amp;auml;ser, SuperFrost Plus</td><td>VWR</td><td>631-9483</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-chicken IgG (H+L)</td><td>Life Technologies</td><td>A11039</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-rabbit IgG (H+L)</td><td>Life Technologies</td><td>A21206</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-rat IgG (H+L)</td><td>Life Technologies</td><td>A21208</td><td></td></tr><tr><td>Alexa Fluor 568 donkey anti-mouse IgG (H+L)</td><td>Life Technologies</td><td>A10037</td><td></td></tr><tr><td>Alexa Fluor 568 donkey anti-rabbit IgG (H+L)</td><td>Life Technologies</td><td>A10042</td><td></td></tr><tr><td>Alexa Fluor 647 goat anti-mouse IgG (H+L)</td><td>Life Technologies</td><td>A21235</td><td></td></tr><tr><td>Anti-5-Bromo-2-Deoxyuridine</td><td>Dako</td><td>M0744</td><td></td></tr><tr><td>Anti-CD140a (PDGFR&amp;alpha;) (rat)</td><td>BD Biosciences</td><td>558774</td><td>Dilute at a ratio 1:500.</td></tr><tr><td>Anti-Chondroitin Sulphate Proteoglycan NG2 (rabbit)</td><td>Merck Milipore</td><td>AB5320</td><td>Dilute at a ratio 1:200.</td></tr><tr><td>Anti-Doublecortin (rabbit)</td><td>Abcam</td><td>ab18723</td><td>Dilute at a ratio 1:200.</td></tr><tr><td>Anti-Doublecortin (chicken)</td><td>Synaptic Systems</td><td>326006</td><td>Dilute at a ratio 1:500.</td></tr><tr><td>Anti-Glial Fibrillary Acidic Protein (rabbit)</td><td>Sigma-Aldrich</td><td>G9269-.2ML</td><td>Dilute at a ratio 1:1000.</td></tr><tr><td>Anti-Myelin Basic Protein (rabbit)</td><td>Cell Signalling Technology</td><td>78896S</td><td>Dilute at a ratio 1:200.</td></tr><tr><td>Anti-Nestin (mouse)</td><td>Merck Milipore</td><td>MAB353</td><td>Dilute at a ratio 1:200.</td></tr><tr><td>Anti-Neuronal Nuclei (mouse)</td><td>Merck Milipore</td><td>MAB377</td><td>Use 6% BSA in PBS 1X. Dilute at a ratio 1:400.</td></tr><tr><td>Anti-SOX2 (rabbit)</td><td>Abcam</td><td>ab97959</td><td>Dilute at a ratio 1:500.</td></tr><tr><td>Anti-Tubulin &amp;beta;3 (rabbit)</td><td>BioLegend</td><td>802001</td><td>Dilute at a ratio 1:200.</td></tr><tr><td>Axiovert 200 wide field microscope</td><td>ZEISS</td><td></td><td></td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>ThermoFisher</td><td>17504044</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B6768-500g</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>NZYTech</td><td>MB04602</td><td></td></tr><tr><td>Cell counting chamber, Neubauer</td><td>Hirschmann</td><td>8100104</td><td></td></tr><tr><td>Cell culture CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>ESCO</td><td>CCL-170B-8</td><td></td></tr><tr><td>Corning Costar TC-Treated 24 Multiple Well Plate</td><td>Corning</td><td>CLS3524-100EA</td><td></td></tr><tr><td>di-Sodium hydrogen phosphate dihydrate</td><td>Merck Milipore</td><td>1.06580.1000</td><td></td></tr><tr><td>DMEM/F-12, GlutaMAX Supplement</td><td>ThermoFisher</td><td>31331028</td><td></td></tr><tr><td>Dumont #5 - Fine Forceps</td><td>FST</td><td>11254-20</td><td></td></tr><tr><td>Dumont #5S Forceps</td><td>FST</td><td>11252-00</td><td></td></tr><tr><td>Dumont #7 Forceps</td><td>FST</td><td>11272-30</td><td></td></tr><tr><td>Epidermal growth factor</td><td>ThermoFisher</td><td>53003018</td><td></td></tr><tr><td>Fibroblast growth factor</td><td>ThermoFisher</td><td>13256029</td><td></td></tr><tr><td>Filter papers</td><td>Whatman</td><td>1001-055</td><td></td></tr><tr><td>Fine Scissors - Sharp</td><td>FST</td><td>14060-09</td><td></td></tr><tr><td>Gillete Platinum 5 blades</td><td>Gillette</td><td></td><td></td></tr><tr><td>HBSS, no calcium, no magnesium</td><td>ThermoFisher</td><td>14175053</td><td></td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>1399</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck Milipore</td><td>1.09057.1000 (1L)</td><td></td></tr><tr><td>Labculture Class II Biological Safety Cabinet</td><td>ESCO</td><td>2012-65727</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>McILWAIN Tissue Chopper</td><td>The Mickle Laboratory Engineering CO. LTD.</td><td>MTC/2</td><td>Set to 450 &amp;mu;m</td></tr><tr><td>Micro Spatula - 12 cm</td><td>FST</td><td>10091-12</td><td></td></tr><tr><td>Micro tube 0.5 mL</td><td>SARSTEDT</td><td>72.699</td><td></td></tr><tr><td>Micro tube 1.5 mL</td><td>SARSTEDT</td><td>72.690.001</td><td></td></tr><tr><td>Micro tube 2.0 mL</td><td>SARSTEDT</td><td>72.691</td><td></td></tr><tr><td>NeuroCult Chemical Dissociation Kit (Mouse)</td><td>Stem Cell</td><td>5707</td><td></td></tr><tr><td>Olympus microscope SZ51</td><td>Olympus</td><td>SZ51</td><td></td></tr><tr><td>Paraformaldehyde, powder</td><td>VWR</td><td>28794.295</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher</td><td>15140122</td><td></td></tr><tr><td>Petri dishes 60 mm</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>Phosphate standard solutions, PO&lt;sub&gt;4&lt;/sub&gt;3 - in water</td><td>BDH ARISTAR</td><td>452232C</td><td></td></tr><tr><td>Poly-D-Lysine 100mg</td><td>Sigma-Aldrich</td><td>P7886</td><td></td></tr><tr><td>Poly-L-ornithine solution</td><td>Sigma-Aldrich</td><td>P4957</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P5405-250g</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>P4170-25MG</td><td></td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>27800.360.5K</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Merck Milipore</td><td>535C549998</td><td></td></tr><tr><td>Triton X-100</td><td>BDH</td><td>14630</td><td></td></tr><tr><td>VWR INCU-Line IL10</td><td>VWR</td><td>390-0384</td><td></td></tr></tbody></table>

isolation and expansion of neurospheres from postnatal (p1&#8722;3) mouse neurogenic niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

SVZ and DG neurospheres, obtained by using the NSA, are composed of undifferentiated cells, positive for Sox2, a transcription factor involved in the self-renewal capacity and positive for nestin, an intermediate filament protein expressed in NSPCs (Figure 1A). In addition, SVZ-derived neurospheres have larger dimensions than their DG counterparts (Figure 1A). Importantly, in differentiative conditions, SVZ- and DG-derived NSPCs migrate out of neurospheres forming a pseudomonolayer of cells (Figure 1B).
To access the self-renewal capacity, the cell pair assay is performed based on the expression of Sox2 and nestin which tends to disappear in dividing cells that start the differentiation process with a combination of a marker of the neuronal lineage namely, DCX. In both neurogenic regions, it is possible to observe the presence of Sox2+/+/nestin+/+/DCX-/- symmetrical divisions (self-renewal) (Figure 2A1,B1), Sox2-/+/nestin-/+/DCX+/- asymmetrical divisions (Figure 2A1,B2) and Sox2-/-/nestin-/-/DCX+/+ symmetrical divisions (differentiation) (Figure 2A2,B1).
Passaging the neurospheres increases the yield of NSPCs; however, cell death at DIV2 changes with passaging. In fact, the percentage of PI-positive cells is increased with cell passage in SVZ (P0: 15.6% &#177; 1.2% vs P1: 19.2% &#177; 2.7% vs P2: 32.35% &#177; 0.14% vs P3: 39.6% &#177; 4.0%) and in DG (P0: 16.31% &#177; 0.95% vs P1: 32.1% &#177; 1.7% vs P2: 27.42% vs P3: 32.2% &#177; 3.1%) (Figure 3).
Neuritogenesis can be evaluated in neurons obtained from the differentiation of SVZ and DG NSPCs at the beginning of differentiation: DIV1 (Figure 4A,D), DIV2 (Figure 4B,E) and DIV3 (Figure 4C,F). In fact, as observed in Figure 4, the length and ramification of the neurites increases with differentiation.
Cell proliferation can be evaluated in SVZ- and DG-derived neurospheres. Comparing primary differentiated neurospheres at DIV1 from SVZ (Figure 5A1) and DG (Figure 5A2), the percentage of BrdU-positive cells is higher in SVZ than in DG (SVZ: 6.15% &#177; 0.64% vs DG: 3.27% &#177; 0.13%; p &#60; 0.05; n = 4; Figure 5A3). Moreover, cell differentiation can also be accessed by combining BrdU staining with a mature maker such as neuronal nuclei (NeuN) that identifies mature neurons (Figure 5B1,B2). Figure 5B3 shows that the percentage of proliferating progenitors that differentiate into mature neurons is similar in SVZ and DG (SVZ: 12.04% &#177; 1.58% vs DG: 13.56% &#177; 0.48%; p &#62; 0.05; n = 4).
The stemness and the multipotency of SVZ- and DG-derived NSPCs can be accessed using the NSA by evaluating the expression of different markers at different differentiation days (DIV2 and DIV7). Indeed, NSCs (nestin- and glial fibrillary acidic protein [GFAP]-double-positive cells) are present in both neurogenic regions (Figure 6A,G). These cells are able to differentiate into immature neurons (DCX-positive cells) (Figure 6B,H), mature neurons (NeuN-positive cells) (Figure 6F,L), oligodendrocyte precursor cells (neuron-glial antigen 2 [NG2] and platelet-derived growth factor receptor &#945; [PDGFR&#945;]- positive cells) (Figure 6C,I), mature oligodendrocytes (myelin basic protein [MBP]-positive cells) (Figure 6E,K) and astrocytes (GFAP-positive cells) (Figure 6D,J).
Different substrates can be used to coat coverslips to form the pseudomonolayer of cells under differentiative conditions. As shown in Supplementary Figure 1, DG cells migrate more when the coverslips have extra-coating with laminin combined with PLO or PDL than with PDL alone (Supplementary Figure 1B&#8722;H). In fact, when PDL and laminin are used together as substrates (Supplementary Figure 1C,G), DG cells form a more confluent pseudomonolayer than SVZ cells for which PDL is used alone (Supplementary Figure 1A,E).
Importantly, these results demonstrate the potential of the NSA to evaluate the stemness and multipotency properties of NSCs derived from the two main neurogenic niches.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60822/60822fig1.jpg" /> 
Figure 1: Subventricular zone and dentate gyrus derived NSPC cultured as neurospheres or as pseudomonolayers. (A) Representative brightfield (A1,A3) and fluorescence (A2,A4) images of SVZ- and DG-derived neurospheres, where nuclei were stained with Hoechst 33342 (blue) and NSCs for Sox2 (green) and nestin (red). (B) Representative brightfield images of pseudomonolayers generated from SVZ- and DG-derived neurospheres under differentiative conditions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60822/60822fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60822/60822fig2.jpg" /> 
Figure 2: The cell pair assay. Representative fluorescence images of cell pairs derived from a progenitor cell division. SVZ and DG nuclei were stained with Hoechst 33342 (blue), stem-like cells for Sox2 (red) and nestin (white) as well as immature neurons with DCX (green). Arrowheads in panels A1 and B1 indicate Sox2+/+/nestin+/+/DCX-/- symmetrical self-renewing divisions, arrows in panels A1 and B2 indicate Sox2+/-/nestin+/-/DCX-/+ asymmetrical divisions, dashed line arrows in panels A2 and B1 show Sox2-/-/nestin-/-/DCX+/+ symmetrical differentiating divisions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60822/60822fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60822/60822fig3.jpg" /> 
Figure 3: Cell survival analysis with cell passaging. Quantitative analysis of PI-positive cells at DIV2 in SVZ- and DG-derived differentiated neurosphere culture, after 0, 1, 2 and 3 passages (P0&#8722;P3). Data is expressed as mean &#177; SEM, n = 1&#8722;8. PI = propidium Iodide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60822/60822fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60822/60822fig4.jpg" /> 
Figure 4: Neuritogenesis analysis at DIV 1, 2 and 3. Representative confocal fluorescence images of neurites, identified by the &#946;III-tubulin signal, in SVZ and DG neurons at (A,D) DIV1, (B,E) DIV2, and (C,F) DIV3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60822/60822fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60822/60822fig5.jpg" /> 
Figure 5: Cell proliferation assay. Representative confocal images of BrdU-positive cells at DIV1 in (A1) SVZ and (A2) DG. (A3) Quantitative analysis of BrdU-positive cells at DIV1 in DG- and SVZ-derived differentiated neurosphere culture. Data is expressed as mean &#177; SEM, n = 4. *p &#60; 0.05 by t-test. Representative fluorescence images of BrdU- and NeuN-positive cells at DIV7 in (B1) SVZ and (B2) DG. Arrowheads indicate BrdU-/NeuN-positive cells. (B3) Quantitative analysis of BrdU-/NeuN-positive cells at DIV7 in both niches. Data is expressed as mean &#177; SEM, n = 4. BrdU: 5-bromo-2&#39;-deoxyuridine, synthetic thymidine analogue. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60822/60822fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60822/60822fig6.jpg" /> 
Figure 6: Neural cell types present in SVZ- and DG-derived differentiated neurosphere culture. Representative fluorescence images of SVZ- and DG-derived cell types after 2 and 7 days of neurosphere differentiation (DIV2 and DIV7), where cell nuclei were stained with Hoechst 33342 (blue) and: (A,G) NSCs for GFAP (green) and nestin (red), (B,H) immature neurons for DCX (green), (C,I) oligodendrocyte precursor cells for PDGFR&#945; (green) and NG2 (red), (D,J) astrocytes for GFAP (green), (E,K) mature oligodendrocytes for MBP (green), and (F,L) mature neurons for NeuN (red). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60822/60822fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/60822/60822suppfig1.jpg" /> 
Supplementary Figure 1: Testing different substrates for neurosphere adherence and migration to form a pseudomonolayer. Representative fluorescence images of (A,E) SVZ-derived pseudomonolayer using poly-D-lysine as a substrate, (B,F) DG-derived pseudomonolayer using poly-D-lysine as a substrate, (C,G) DG-derived pseudomonolayer using poly-D-lysine with laminin as a substrate, and (D,H) DG-derived pseudomonolayer using poly-D-lysine with poly-L-ornithine as a substrate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60822/60822suppfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Expansion of Neurospheres from Postnatal P1âˆ'3 Mouse Neurogenic Niches at JoVE.com

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

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Nanyang Technological University

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JoVE Journal

Neuroscience

14.2K Views.  Universidade de Lisboa. De neurosfeertest is nuttig in vitro techniek voor het bestuderen van de inherente eigenschappen van neurale stam / voorloper cellen met inbegrip van proliferatie, zelfvernieuwing en multipotentie in zowel fysiologische en pathologische context. Door zijn driedimensionale structuur is deze techniek een krachtig middel voor het bestuderen van volwassen neurogenese. Het is ook nuttig voor het kweken en het verkrijgen van hogere opbrengsten van neurale stam / voorloper cellen.Deze neurosf...

Video: Isolation and Expansion of Neurospheres from Postnatal (P1−3) Mouse Neurogenic Niches

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 ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

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 ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

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

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

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

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

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

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

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

Isolation and Culture of Embryonic Mouse Neural Stem Cells

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

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

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

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 ...

Formation and Expansion of Neurospheres from a Hippocampal Tissue

Source: Soares, R. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60822">Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches.</a>&#160;J. Vis. Exp. (2020).
This video demonstrates the process of neurosphere formation and their expansion from hippocampal tissue. Neural stem and progenitor cells from the postnatal mouse dentate gyrus tissue proliferate and form primary neurospheres, which are then reseeded to form secondary neurospheres.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Basic ...

Isolation and Expansion of Neurospheres from Postnatal (P1−3) Mouse Neurogenic Niches

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