Name: isolation and expansion of neurospheres from postnatal (p1&#8722;3) mouse neurogenic niches
Uploaded: 2020-05-23T13:00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Isolation and Expansion of Neurospheres from Postnatal P1Ã¢Ë†'3 Mouse Neurogenic Niches at JoVE.com

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.

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;-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 &#38; C&#170; (Irm&#227;o) Sucrs, Lda</td>
			<td>UN1170</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion slides, Menzel Gl&#228;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></td>
			<td></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&#945;) (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 &#946;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 CO2 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 &#956;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, PO43 - 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/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/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/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/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/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/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/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 P1Ã¢Ë†'3 Mouse Neurogenic Niches at JoVE.com

Isolation and Expansion of Neurospheres from Postnatal P1ÃƒÂ¢Ã‹â€ '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.

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 ...

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