This work was supported by funding from the Overstreet Foundation.

1. Basic Set Up Before Proceeding to Cell Culture
<ol>
 <li>NeuroCult NSC Basal Medium and NeuroCult NSC Proliferation Supplements are mixed at a 9:1 ratio, respectively, to make complete NSC medium.</li>
 <li>A 37 &deg;C water bath is used to warm up the medium.</li>
 <li>Appropriate amount of heat inactivated fetal calf serum (FCS) is thawed.</li>
 <li>Appropriate amount of stock growth factor solutions including epidermal growth factor (EGF at 10 &mu;g/ml), basic fibroblastic growth factor (b-FGF at 10 &mu;g/ml) and 0.2% heparin solution in phosphate buffered saline (PBS) are thawed.</li>
 <li>Tissue culture flasks (T25, T75 or &amp;T175) are needed for NSC expansion and differentiation.</li>
 <li>Tissue culture treated 96 and 24 well plates and glass coverslips are needed for plating sorted cells.</li>
</ol>
2. Isolation, and Expansion (Passaging) of NSCs
<ol>
 <li>Neural stem and progenitors are isolated and expanded from mouse brain as described in separate protocols 1, 2 3.</li>
 <li>When the neurospheres (either primary or passaged neurospheres) are ready for subculture (150-200 &mu;m in diameter), the medium containing spheres is transferred to an appropriate size sterile tissue culture tube, and centrifuged at 800 rpm (110 g) for 5 min at room temperature.</li>
 <li>The supernatant medium is removed and the spheres are re-suspended in 1 ml of pre-warmed 0.05 % trypsin-EDTA.</li>
 <li>After incubation in a 37 &deg;C water bath for 2-3 min, an equal volume of soybean trypsin inhibitor is added to the cell suspension to stop the trypsin activity.</li>
 <li>To ensure that the trypsin has been completely inactivated, the cell suspension is gently pipetted up and down 3-5 times. </li>
 <li>After centrifugation at 800 rpm (110 g) for 5 min, the supernatant is removed and the cells are re-suspended in 1 ml of complete NSC medium. </li>
 <li>10&mu;l of the cell suspension is mixed with 90 &mu;l of trypan blue to perform a cell count. 
 Note: Other appropriate cell dilutions can also be used. 
 </li>
 <li>The cells are then used for subculture 1, 2 or for differentiation as described below.</li>
</ol>



3. Differentiation of Neural Stem and Progenitor Cells, The Neuroblast Assay (NBA)
<ol>
 <li>Cells resulting from dissociated neurospheres are plated at a concentration of 2-3 x 105 cells/ml in the complete NSC medium supplemented with 20 ng/ml EGF, 10 ng/ml bFGF, 1 &mu;l/ml of 0.2% heparin and 5% heat inactivated FCS in an appropriate size tissue culture flask. 5 ml medium is used for T25, 20 ml for T75 and 40 ml for T175 flasks. Note: FCS will cause the cells to firmly attach to the substrate and there is no need for coating the flasks for NBA culture. 
 </li>
 <li>Culture flasks are then incubated in a 37 &deg;C humidified incubator with 5% CO2 for 3-4 days. This period is called proliferation stage, during which NSC progeny attach to the substrate and proliferate as a monolayer of cells.</li>
 <li>When the culture becomes 90-95% confluent, the medium of each flask is switched to the complete NSC medium supplemented with 5% FCS without any growth factors.</li>
 <li>Culture flasks are again incubated in a 37 &deg; C humidified incubator with 5% CO2 for another 3-4 days. This period is called differentiation stage, during which neuronal progenitors appear on top of an astrocytic monolayer and proliferate to make colonies of immature neuronal cells. On day 4-5 of differentiation stage, the culture is ready for isolation of the neuroblast cells using flow cytometry technology.</li>
</ol>
4. Cell Preparation for Flow Cytometry
Trypsinization
<ol>
 <li>The culture of differentiating NSC progeny on day 4-5 of differentiation stage is washed once with an appropriate amount of PBS (2 ml for T25, 4 ml for T75 and 8 ml for T175 flask) to remove FCS from the culture. 
 Note: FCS blocks Trypsin-EDTA activity. 
 </li>
 <li>Then, a sufficient amount of pre-warmed 0.05% Trypsin-EDTA (2 ml for T25, 4 ml for T75 and 8 ml for T175 flask) is added to each flask to cover the cell monolayer and incubated for 1-2 min in a 37 &deg;C humidified incubator.</li>
 <li>The flask is checked under the microscope to evaluate the effect of Trypsin-EDTA on the culture. The flask is then held with one hand and struck firmly with the other hand to dislodge the cells from the bottom of the flask.</li>
 <li>Equal volume of soybean trypsin inhibitor is added to the flask to quench trypsin activity. The cell suspension is gently pipetted up and down to ensure trypsin inactivation and to achieve a homogenous single cell suspension. 
 Note: Alternatively, medium supplemented with 5% FCS could be used to quench trypsin activity. 
 </li>
 <li>To remove non-dissociated clumps, the cell suspension is passed through a 40-&mu;m-size mesh filter.</li>
 <li>The cell suspension is then centrifuged at 800 rpm (110g) for 5 min, the supernatant is removed and the cells are re-suspended in an appropriate volume of complete NSC medium. </li>
 <li>The final volume of cell suspension is adjusted so that the cell density does not exceed 2-3x106-cells/ml. High cell density might cause stream blockage in the flow cytometry machine.</li>
 <li>If you would like to isolate neuronal cells just based on their physical properties, now you can proceed to FACS sorting as described in part 4.</li>
 <li>Before sorting, Propidium Iodide (PI, Sigma, 500 &mu;g/ml in PBS) is added at a concentration of 1 &mu;l/ ml of cell suspension to exclude dead cells.</li>
</ol>
Live cell immunolabeling
To achieve a higher purity of neuronal cells, harvested single cell from the NBA culture can be stained for PSA-NCAM (a marker of early immature neuronal progenitors) and then the positive cells from the neuronal population can be isolated using the FACS machine.
<ol>
 <li>After performing a cell count, the single cell suspension from the NBA culture is divided into four groups:
 <ul>
 <li>Cells alone group, serves as a control to adjust parameters and gates (0.5-1 x 106 cells).</li>
 <li>Cells plus PI, serves as a control to adjust parameters and gates (0.5-10 x 106 cells). PI negative cells from this group can also be sorted based on the cells physical characteristics.</li>
 <li>Secondary alone (Isotype control) group that also serves as a control to adjust parameters and gates (1-2 x 106cells).</li>
 <li>Immunolabeling group (8-10 x 106 cells) that is stained for PSA-NCAM to achieve further purification of the immature neuronal cells. To exclude dead cells upon sorting, PI is added to this group as well.</li>
 </ul> 
 </li>
 <li>To start immunolabeling, cells are centrifuged at 800 rpm (110 g) for 5 min. The supernatant is removed and the cells are re-suspended in 100 &mu;l of complete NSC medium.</li>
 <li>Phycoerythrin (PE) conjugated anti-PSA-NCAM antibody is added at a ratio of 10 &mu;l / 5-10 x 106 cells to the cell suspension to be stained for PSA-NCAM. In the same way, a PE conjugated mouse IgG1 isotype control antibody is added to the isotype control cell suspension. Follow the instructions on the antibody specification sheet regarding the concentration of antibody to be used. The cell suspension is then mixed gently and incubated at room temperature for 10-15 min in dark. </li>
 <li>The cell suspensions (PSA-NCAM and Isotype control tubes) are then centrifuged at 800 rpm (110 g) for 5 min, the supernatant is removed and the cells are re-suspended in 1 ml of complete NSC medium.</li>
 <li>To remove excess un-bounded antibodies from the samples, step 4 is repeated 2-3 times.</li>
 <li>After re-suspending cells in an appropriate volume of medium, Propidium Iodide (PI, Sigma, 500 &mu;g/ml in PBS) is added at a concentration of 1 &mu;l/ml of the cell suspensions (cells plus PI, isotype control and PSA-NCAM-stained samples) to exclude dead cells when analyzed by flow cytometry.</li>
 <li>15 ml sterile falcon tubes containing 1 ml of complete medium are prepared to collect sorted cells from different cell populations. </li>
</ol>



5. Fluorescence-activated Cell Sorting (FACS) of Immature Neurons
<ol>
 <li>The FACS machine is set up based on its user manual instruction via the flow cytometry facility expert technician. </li>
 <li>Phosphate buffered saline (PBS) or any other appropriate solution depending on manufacturer instructions, could be used as the sheath fluid at 28 PSI through a 90 &mu;m nozzle. </li>
 <li>Differential pressure on the system is adjusted such that the sort trigger rate does not exceed 2500 events/second. </li>
 <li>The cell suspension from the cells alone group is run through the FACS machine. </li>
 <li>First, the cells (events) are plotted based on their size (Forward Scatter (FSC) light) versus internal complexity (Side Scatter (SSC) light) to distinguish different cell populations. To do so, voltage parameters for FSC and SSC should be adjusted so that the cells can be seen in the flow cytometry plot.</li>
 <li>To exclude cell clumps and doublets, the cells are first plotted based on forward scatter area (FSC-A) versus forward scatter pulse width (FSC-W) and the single cell population is gated as population 1 (P1). Then the P1 cells are plotted based on side scatter area (SSC-A) versus side scatter pulse width (FSC-W) and this time the single cells are gated as population 2 (P2). Having established these two consecutive gates, clumps or doublets (high FSC pulse width and high SSC pulse width) are being excluded.</li>
 <li>To exclude dead or damaged cells, the single cells (P2) are plotted based on PI reactivity versus FSC-A and the single live cell population is gated. </li>
 <li>Single live cells are plotted based on FSC versus SSC to distinguish different cell populations based on cell size and internal granularity. At this stage two main cell populations are gated as FSClow SSClow (P3) and FSChigh SSChigh (P4) cell populations. </li>
 <li>To sort PSA-NCAM immuno-reactive immature neuronal cells from the FSClow SSClow population, first the FSClow SSClow population from control (cells plus PI and isotype control) groups is plotted based on PE immunoreactivity versus FSC-A and the negative cells are gated (P5), and then positive cells from PSA-NCAM stained group are gated as population 6 (P6). </li>
 <li>After adjusting all needed gates, cells of interests are sorted into 15 ml sterile tissue culture tubes containing 1ml NSC medium. </li>
 <li>Sorted cells are then centrifuged at 1200 rpm (240 g) for 5 min.</li>
 <li>Supernatant is discarded and the pellet is re-suspended in an appropriate volume of medium (depending on the pellet size) and a cell count is performed.</li>
 <li>Cells are then plated in Poly-L- Ornithine coated 96-well plates at a density of 20-30 x 103 cells /well in 200-250 &mu;l of complete NSC medium with 5% FCS and 20 ng/ml human recombinant BMP4 5.</li>
</ol>
 Note: to coat 96 wells, 100 &mu;l of Poly-L- Ornithine working solution (1.5 ml Poly-O and 8.5 ml of sterile PBS) is added to each well and the plate is incubated in a 37 &deg;C incubator for at least one hour. Then, the Poly-O is removed and each well is washed three times with sterile PBS (let the PBS remain in the well for 10 min each time). Use 500 &mu;l of Poly-L- Ornithine working solution / well if 24-well plates are used. 
 <ol start="14">
 <li>Cells are then fixed at different time points after sort using cold 4% paraformaldehyde and immunostained for appropriate neuronal and glial cell markers to assess the phenotype of sorted cells from different cell populations.
 </li>
</ol>
6. Representative Results
In the neuroblast assay (NBA) differentiation culture, plated cells attach to the tissue culture substrate and grow as a monolayer. Depending on initial cell plating density, the culture will turn into a 90-95% confluent culture after 3-4 days. At this stage, a monolayer of mainly astrocytic cells can be visualized underneath with small round neuronal progenitor cells on top4,5 (Figure 1). After switching the medium to a growth factor free medium, neuronal progenitor cells start dividing rapidly and generate clusters of immature neuroblast cells on top of the astrocytic monolayer in 4-5 days (Figure 2). Flow cytometry analysis of dissociated NBA culture on day 4-5 of differentiation stage, shows two main cell populations; FSClow SSClow and FSChigh SSChigh based on cell size (FSC) and internal granularity (SSC) (Figure 3). Immuno-fluorescent analysis of the sorted cells shows that the neuronal cells are mainly located in the FSClow SSClow populations (with more than 75% of them expressing &beta;-III tubulin), while sorted cells from the FSChigh SSChigh population are mostly GFAP immunoreactive astrocytes (Figure 4). Further purification of immature neuronal cells up to near homogeneity (97%) could be achieved with positive selection of PSA-NCAM IR cells from the FSClow SSClow population (Figure 3, 4). Enriched neuronal cells generated from the NSCs isolated from E14 mouse ganglionic eminences acquire a GABAergic phenotype and express DARPP-32, a marker of medium spiny neurons, upon differentiation (Figure 5).
<img src="/files/ftp_upload/3712/3712fig1.jpg" alt="Figure 1"/> 
Figure 1. Neuroblast assay culture from E14 mouse neural stem cells on day 4 of proliferation stage. This monolayer culture consists of flat astrocytic cells (arrowheads) underneath and round neuronal progenitor cells (arrows) on top. Original magnification; 20 x.
<img src="/files/ftp_upload/3712/3712fig2.jpg" alt="Figure 2"/> 
Figure 2. Neuroblast assay culture from E14 mouse neural stem cells on day 4 of differentiation stage: A) Phase contrast, B) Immunofluorescent staining. Note the cluster of immature neuronal cells (arrows in A and &beta; III tubulin staining in B) on top of the astrocytic monolayer (arrow heads in A, GFAP staining in B). Original magnification; 20 x.
<img src="/files/ftp_upload/3712/3712fig3.jpg" alt="Figure 3"/> 
Figure 3. Representative sort plots: A). FSC vs. SSC sort plot before exclusion of doublets and dead cells. B, C). Sort plots for exclusion of doublets based on FSC and SSC pulse width. D). Exclusion of dead and damaged cells based on Propidium Iodide immunoreactivity. E). FSC vs. SSC plot after exclusion of doublets and dead cells, showing the two main populations labeled as the FSClow SSClow and the FSChigh SSChigh populations. F, G). Sort plots to isolate PSA-NCAM IR immature cells from the FSClow SSClow population.
<img src="/files/ftp_upload/3712/3712fig4.jpg" alt="Figure 4"/> 
Figure 4. Representative micrographs from cells sorted from different populations one day after plating. A) FSClow SSClow population (more than 75% of these cells are neurons). B) FSChigh SSChigh population (the majority of these cells are astrocytes). C) PSA-NCAM+ sorted cells from FSClow SSClow population (almost all of the sorted cells are neurons). Original Magnification; 20 x.
<img src="/files/ftp_upload/3712/3712fig5.jpg" alt="Figure 5"/> 
Figure 5. Sorted immature neurons differentiate into GABAergic neurons (A) and express DARPP-32 (B), a marker for medium spiny neurons.

<ol>
	<li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M., Ansari, S., Reynolds, B. <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> J. Vis. Exp. (47), e2457 (2011).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <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+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods. Mol. Biol. 750, 61-77 (2011).</li><li>Scheffler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+characterization+of+adult+brain+neuropoiesis.">Phenotypic and functional characterization of adult brain neuropoiesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 9353-9358 (2005).</li><li>Azari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+immature+neuronal+cells+from+neural+stem+cell+progeny.">Purification of immature neuronal cells from neural stem cell progeny.</a> PLoS One. 6, e20941-e20941 (2011).</li><li>Amariglio, N. <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 Medicine. 6, 221-231 (2009).</li></ol>

The authors have nothing to disclose.

The ability to isolate defined neural cell populations (i.e. neurons, astrocytes and oligodendrocytes) might solve some of the impediments associated with the clinical application of neural stem cells (NSCs). First, it enables us to fully characterize the starting population of donor cells. Second, it allows us to use a particular cell type or a combination of different cell types with a specific ratio, depending on the nature or stage of the disease. Lastly, using highly enriched pre-differentiated neural cell progenito...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>NeuroCult NSC Basal Medium</td>
 <td>Medium</td>
 <td>Stem Cell Technologies</td>
 <td>05700</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NeuroCult NSC Proliferation Supplements</td>
 <td>Medium supplement</td>
 <td>Stem Cell Technologies</td>
 <td>05701</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>%0.05 &nbsp;&nbsp; trypsin-EDTA</td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>25300-062</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Soybean trypsin inhibitor </td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pen/Strep </td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>15140-122</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>*MEM</td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>41500-018</td>
 <td>HEM component</td>
 </tr>
 <tr>
 <td>*HEPES</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>H4034</td>
 <td>HEM component</td>
 </tr>
 <tr>
 <td>*Distilled water</td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>15230-147</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell strainer </td>
 <td>Sieve</td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>T25 flask</td>
 <td>Culture ware</td>
 <td>Nalge Nunc international</td>
 <td>136196</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>T80 flask</td>
 <td>Culture ware</td>
 <td>Nalge Nunc international</td>
 <td>178905</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml tubes</td>
 <td>Culture ware</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml tubes</td>
 <td>Culture ware</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>Growth factor</td>
 <td>R&amp;D</td>
 <td>2028-EG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>b-FGF</td>
 <td>Growth factor</td>
 <td>R&amp;D</td>
 <td>3139-FB</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>Growth factor</td>
 <td>Sigma</td>
 <td>H4784</td>
 <td>Reconstituted in PBS</td>
 </tr>
 <tr>
 <td>Anti-PSA-NCAM-PE</td>
 <td>Antibody</td>
 <td>Miltenyi Biotec</td>
 <td>130-093-274</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PE- Conjugated mouse IgG1</td>
 <td>Isotype control antibody</td>
 <td>BD Pharminogen</td>
 <td>556029</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>Growth factor</td>
 <td>Sigma</td>
 <td>H4784</td>
 <td>Reconstituted in PBS</td>
 </tr>
 <tr>
 <td>HrBMP-4</td>
 <td>Growth factor</td>
 <td>R&amp;D</td>
 <td>314-BP-010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Poly-L-Ornithine</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>P4957</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Calf Serum</td>
 <td>Medium Supplement</td>
 <td>Gibco</td>
 <td>10099-141</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GFAP</td>
 <td>Antibody</td>
 <td>DAKO </td>
 <td>Z0334</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>B-III tubulin</td>
 <td>Antibody</td>
 <td>Promega</td>
 <td>G7121</td>
 <td>&nbsp;</td>
 </tr>
</table>
*To make HEM, mix 1 x 10 L packet of MEM and 160 ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4 &deg;C.

the neuroblast assay: an assay for the generation and enrichment of neuronal progenitor cells from differentiating neural stem cell progeny using flow cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

Watch this Scientific Journal Video about The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytomet

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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15.4K Views.  The University of Florida. This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Video: The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

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 ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

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. ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...