This work was supported by funding from the Overstreet Foundation.

1. Items That Need to Be Prepared Before Proceeding to Cell Plating:
<ol>
	<li>Appropriate volume of complete NSC medium is prepared by mixing NeuroCult NSC Basal Medium and NeuroCult NSC Proliferation Supplements at a 9:1 ratio, respectively.</li>
	<li>An aliquot of NeuroCult NCFC Serum-Free Medium without Cytokines is thawed.</li>
	<li>The medium is warmed up in a 37&deg;C water bath.</li>
	<li>Stock solutions of epidermal growth factor (EGF), basic fibroblastic growth factor (b-FGF) at the concentration of 10 &mu;g/mL and heparin at 0.2% are prepared ahead.</li>
	<li>Depending on the experiment size, several 35mm tissue culture dishes are needed to plate the cells and one 150-200cm plastic Petri dish is also needed to hold the duplicate 35mm dishes and a third 35mm dish for water.</li>
</ol>
2. Cell Preparation:
Depending on your experiment, cells can be prepared from an adult or embryonic source (primary dissociated tissue or dissociated neurospheres). Dissect tissues from adult/embryonic mouse central nervous system (CNS) or dissociate adult/embryonic-derived neurospheres as described before 1,2 and then:
<ol>
	<li>The single cell suspension is passed through a 40-&mu;m-size mesh filter so as to remove non-dissociated clumps.</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>If using primary embryonic or adult derived neural cells, dilute the cell suspension to a concentration of 6.5 x 105 cells/mL in complete NSC medium. If using cells from dissociated neurospheres derived from adult or embryonic neural cells, dilute the cell suspension to a concentration of 2.2 x 105 cells/mL in complete NSC medium.</li>
</ol>
3. Plating Cells in Semi-solid N-CFCA Medium:
<ol>
	<li>The appropriate volume of the following components is mixed in order, depending on the number of replicates. Here we mix the amount needed for two replicates or duplicate dishes. For additional numbers of replicates, please refer to table 1.
	<ul>
		<li>1.7 mL of NeuroCult NCFC Serum-Free Medium without Cytokines</li>
		<li>330 &mu;L of NeuroCult NSC Proliferation Supplements</li>
		<li>6.6 &mu;L of EGF (10&mu;g/mL)</li>
		<li>32 &mu;L of Penicillin/Streptomycin</li>
		<li>3.3 &mu;L of b-FGF (10&mu;g/mL) required only if culturing cells derived from adult neural cells.</li>
		<li>3.3 &mu;L of heparin (0.2%) required only if culturing cells derived from adult neural cells.</li>
		<li>25&mu;L of cell suspension (of 2.2 x 105 cells/mL cells from dissociated neurospheres or 6.5 x 105 cells/mL primary cells from dissociated CNS tissue). Note: The final cell numbers plated should be about 2500 cells per 35 mm culture dish for neurosphere derived cells and 7500 cells per 35 mm culture dish for primary cells. In some cases, the final cell plating density has to be adjusted by performing a cell titration experiment. For statistical analyses, the total number of colonies detected after 21 days of culture should be within the range of at least 50 - 150 colonies.</li>
	</ul>
	</li>
	<li>The medium containing the cells is mixed gently and then 1.3 mL of cold Collagen solution is transferred to the cell suspension and mixed well by gentle pipetting to avoid introducing any bubbles.</li>
	<li>The mixed solution is dispensed to the center of each 35mm culture dish (~1.5 mL/dish) and the dishes are tipped gently using a circular motion to let the mixture spread evenly over the surface of the dish. .</li>
	<li>The duplicate 35 mm culture dishes are placed into a 100 mm Petri plate. The lid of new 35 mm dish is removed and the open dish is also placed in the same 100 mm Petri dish. Sterile water is added to this open 35 mm dish to maintain optimal humidity during the incubation period. Square bioassay plates (245 mm) are used when more replicate 35 mm dishes have been set up. Again, include 2 or 3 open 35 mm dishes containing sterile water.</li>
	<li>The plates are transferred to an incubator set at 37&deg;C, 5% CO2 and 95% humidity. The collagen congeals by increasing temperature and gel formation will occur within approximately one hour. The cultures should not be disturbed during this time.</li>
	<li>Cultured cells are incubated for 21 days (differences in colony size can be clearly distinguished after 21 days).</li>
</ol>
4. Preparing Replenishment Medium and Feeding the Culture:
As N-CFCA cultures are incubated for an extended period of time (21 days), cultures should be fed with the appropriate complete NeuroCult replenishment medium prepared fresh as follows:
<ul>
	<li>4.5 mL of NeuroCult NSC Basal Medium is mixed with 0.5 mL of NeuroCult NSC Proliferation Supplements and then the growth factors is added:</li>
	<li>250 &mu;L of 10&mu;g/mL EGF (to give a final concentration of 0.5 &mu;g/mL EGF)</li>
	<li>125 &mu;L of 10&mu;g/mL b-FGF (to give a final concentration of 0.25&mu;g/mL b-FGF) required only if culturing cells derived from adult neural cells.</li>
	<li>125 &mu;L of 0.2% Heparin, required only if culturing cells derived from adult neural cells.</li>
	<li>60 &mu;L of the appropriate complete NeuroCult Replenishment Medium (for embryonic or adult cells) is added into the center of each NCFC Assay dish once every 7 days during the entire NCFC Assay culture incubation (21 days).</li>
</ul>
5. Scoring N-CFC Assay Derived Colonies and Calculating the Frequency of bona Fide NSCs and Neural Progenitor Cells:
<ul>
	<li>Each 35 mm culture dish is placed on a gridded scoring dish with the grid size of 2.0 mm x 2.0 mm and then both dishes are placed on the microscope stage.</li>
	<li>Using low power (2.5X - 5X) objective lens, each dish is scanned and the colonies are scored based on their sizes.</li>
	<li>Colonies are classified into two major categories:
	<ol>
		<li>Less than 2 mm diameter</li>
		<li>&ge;2 mm diameter</li>
	</ol>
	</li>
</ul>
6. Representative Results:
As in neurosphere assay, cells plated in N-CFC assay start to proliferate and make small colonies of cells within 3 - 7 days after plating (Figure 1). In two weeks time, colonies of different sizes can be distinguished. While the majority of colonies tend to stop growing after 14 days, some colonies continue to increase in size. By day 21, colonies can be classified into one of the four categories: 1) less than 0.5 mm diameter, 2) 0.5 - 1 mm diameter, 3) 1 - 2 mm diameter and 4) &ge;2mm in diameter. Practically, colonies smaller than 2 mm diameter are referred to as progenitor derived (Figure 2) and colonies &ge;2mm in diameter are referred to as NSC derived (Figure 3). The number of colonies &ge;2mm in diameter per total cells plated, represents the frequency of the actual bona fide neural stem cells with long-term self-renewal and multi-potential capabilities. The total neurosphere forming frequency in a particular cell population after 7-8 day in a parallel NSA experiment has been estimated to be similar to the total colony forming frequency of the same cell population after 21 days in a N-CFCA experiment. The N-CFCA however, provides a more permissive condition so as each cell can show its full proliferative potential.
<table border="1">
<tbody>
 <tr>
 <td>Component</td>
 <td>2 replicates</td>
 <td>3 replicates</td>
 <td>4 replicates</td>
 </tr>
 <tr>
 <td>NeuroCult NCFC Serum-Free Medium without Cytokines </td>
 <td>1700</td>
 <td>2550</td>
 <td>3400</td>
 </tr>
 <tr>
 <td>NeuroCult NSC Proliferation Supplements</td>
 <td>330</td>
 <td>495</td>
 <td>660</td>
 </tr>
 <tr>
 <td>EGF (10 &mu;g/mL) </td>
 <td>6.6</td>
 <td>9.9</td>
 <td>13.2</td>
 </tr>
 <tr>
 <td>bFGF (10 &mu;g/mL), only for adult cells</td>
 <td>3.3</td>
 <td>4.95</td>
 <td>6.6</td>
 </tr>
 <tr>
 <td>Heparin Solution (0.2%), only for adult cells</td>
 <td>3.3</td>
 <td>4.95</td>
 <td>6.6</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin (1:100)</td>
 <td>32</td>
 <td>48</td>
 <td>64</td>
 </tr>
 <tr>
 <td>Cells at: 
 2.2 x 105 cultured cells/mL OR 
 6.5 x 105 primary cells/mL </td>
 <td>25</td>
 <td>37.5</td>
 <td>50</td>
 </tr>
 <tr>
 <td>Collagen Solution </td>
 <td>1300</td>
 <td>1950</td>
 <td>2600</td>
 </tr>
</tbody>
</table>
Table 1. Components of complete N-CFC assay culture.
<img src="/files/ftp_upload/2639/2639fig1.jpg" alt="Figure 1" /> 
Figure 1. Representative colonies in N-CFCA culture of passage one E14 mouse NSCs 7 days after plating. The colonies might display different morphology and size. Original magnification; 4x 
<img src="/files/ftp_upload/2639/2639fig2.jpg" alt="Figure 2" /> 
Figure 2. Representative progenitor derived colonies of different sizes in N-CFCA culture of passage one E14 mouse NSCs 21 days after plating. As shown, the colonies have different morphology but all are less 2 mm in size. Original magnification; 4x 
<img src="/files/ftp_upload/2639/2639fig3.jpg" alt="Figure 3" /> 
Figure 3. Representative bona fide stem cell derived colony in N-CFCA culture of passage one E14 mouse NSCs 21 days after plating. Stem cell derived colonies might display different morphology (see the video) but all are &#8805;2mm in diameter. Original magnification; 4x

<ol>
	<li>Azari, H., Rahaman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J Vis Exp. , (2010).</li><li>Azari, H., Sharififar, S., Rahaman, 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> J Vis Exp. , (2011).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Reynolds, B. A., Rietze, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+Neural+Stem+and+Progenitor+Cells+in+the+Neural+Colony+Forming+Cell+Assay.">Enumeration of Neural Stem and Progenitor Cells in the Neural Colony Forming Cell Assay.</a> Stem Cells. , (2008).</li><li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26, 979-987 (2008).</li><li>Louis, S. A., 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=Neurosphere+and+Neural+Colony-Forming+Cell+Assays.">Neurosphere and Neural Colony-Forming Cell Assays.</a> Protocols for Neural Cell Culture. 10, 1-28 (2010).</li></ol>

One of the authors, Sharon A. Louis, is affiliated with StemCell Technologies Inc, a producer of some reagents used in this study.

Although neurosphere assay3,1,2 is the most common method to isolate and expand neural stem cells from a variety of sources like adult and embryonic CNS tissue, it cannot accurately measure the NSC frequency in a mixed population of neural precursor cells (stem and progenitors) as there is not a one to one relationship between the number of neurospheres and the number of bona fide stem cells4. To address this limitation, the original NSA has been adapted so as to allow the neural stem and progenitors to grow to their full proliferation capacity for three weeks 5-7. Unlike the liquid NSA, in the N-CFCA the colonies are actually clonally derived, as the semi-solid collagen matrix prevents migration of the single cells plated and aggregation of colonies. For consistent results with this assay we recommend:
<ol>
	<li>Ensure a single cell suspension in the original cell suspension. Pass your single cell suspension through a 40-&mu;m-size mesh filter so as to remove non-dissociated clumps.</li>
	<li>Keep the collagen solution on ice or in the +4&deg; C fridge. Collagen is the last item to be added to the mixture of cell suspension as it congeals by increasing the temperature.</li>
	<li>Do not forget to feed the culture every week. Adding the replenishment medium containing growth factor(s) once every 7 days will allow cells to continue to proliferate over the extended culture period.</li>
<li>The final cell plating density have been optimized for cells derived from either primary or cultured neural cells so that the total number of colonies detected after 21 days of culture in the NCFC-A is within the range of at least 50 - 200 colonies. This range allows detection of the rare NSC derived colonies &gt;2mm in diameter in samples while providing sufficient numbers of colonies for statistical analyses. Depending on the starting cell source, significantly fewer (&lt;50) and higher numbers of colonies (&gt;250) are sometimes obtained. Too few colonies will result in the colonies &gt;2mm in diameter being below detection levels, while too many colonies will result in overcrowding anddepletion of growth medium components resulting in an inaccurate colony counting. Therefore cell plating density will need to be adjusted accordingly (i.e. increasing or decreasing the total number of cells plated)</li>
</ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NeuroCult NSC Basal Medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05700</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>STEMCELL</td>
<td>05701</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NCFC medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05720</td>
<td>&nbsp;</td>
<tr>
<td>0.05% 	trypsin-EDTA</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25300-062</td>
<td>&nbsp;</td>
<tr>
<td>Soybean trypsin inhibitor </td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6522</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer </td>
<td>Sieve</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>T25 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>136196</td>
<td>&nbsp;</td>
<tr>
<td>T80 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>178905</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>Reagent </td>
<td>STEMCELL</td>
<td>04902</td>
<td>&nbsp;</td>
<tr>
<td>EGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>2028-EG</td>
<td>&nbsp;</td>
<tr>
<td>b-FGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>3139-FB</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>Growth factor</td>
<td>Sigma</td>
<td>H4784</td>
<td>Reconstituted in PBS</td>
</tr>
<tr>
<td>15 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>35 mm culture dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27100</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27500</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells at JoVE.com

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

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). ...

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22.0K Views.  University of Florida. 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.

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

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 ...

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 ...

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.

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 ...

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 ...

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. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

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 ...

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 ...

Methods Article

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