This work was supported by funding from the Overstreet Foundation.

Part 1: Basic set up before proceeding to dissection:
<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. NSC medium can also be made in laboratory based on NSC growth medium composition published in the literature1. If your lab does not have much experience with making medium, we strongly recommend to use commercially available medium that has been quality controled for NSCs growth prior to being sold (which is the case for NeuroCult, Stem Cell Technolgies).</li>
	<li>The medium is warmed up in a 37&deg;C water bath.</li>
	<li>Cold HEPES-buffered minimum essential medium (HEM) with high concentration of antibiotics (10%) is prepared for dissection and washing purpose. Alternatively, NSC basal medium with antibiotics supplementation may also be used for this purpose. </li>
	<li>30-40 ml of cold HEM containing antibiotics is dispensed into sterile 50 ml tubes for brain collection.</li>
	<li>Several 10cm plastic Petri dishes are needed to hold the brains during dissection and one 10cm sterile glass Petri dish to hold dissected tissue.</li>
	<li>The surgical tools needed to remove the brain (large scissors, small pointed scissors, large forceps, small curved forceps, and a small spatula) or for tissue dissection (small forceps, curved fine forceps, and scalpel) are sterilized using glass bead sterilizer at 250&deg;C or other available autoclave methods.</li>
	<li>Dissection microscope is wiped with 70% alcohol and set up inside the PC2 hood.</li>
</ol>
Part 2: Harvesting adult mouse brain and micro-dissection:
<ol>
	<li>2-4 adult (5-8 weeks old) mice are anesthetized according to one's institutional approved animal protocol using 3-4% isoflurane or by intra-peritoneal injection of pentobarbital (120 mg/kg).</li>
	<li>Cervical dislocation is performed to make sure the animal does not suffer pain and distress.</li>
	<li>The anesthetized mouse is laid on its abdomen on an absorbent tissue paper, and the head is rinsed with 70% ethanol to sterilize the area.</li>
	<li>Large scissors are used to decapitate the animal just above the cervical spinal cord region.</li>
	<li>Small pointed scissors are used to make a median caudal-rostral cut and to expose the skull.</li>
	<li>The cut edges of the skin are used to hold the head and then each blade of a small scissors is placed in each orbital cavity to make a coronal cut between the orbits.</li>
	<li>Using the foramen magnum as an entry point, a longitudinal cut is made through the skull along the sagittal suture and two lateral cuts are also made at the junction of the lateral walls and the base of skull. Caution should be taken not to damage the underlying brain by making small cuts and ensuring the angle of the blades is as shallow as possible.</li>
	<li>The skull overlying each hemisphere is grasped and peeled outward using a curved forceps to expose the brain, then using a small wetted spatula, the brain is scooped into a 50 ml tube containing HEM.</li>
	<li>This procedure is repeated until all of the brains have been harvested.</li>
	<li>The brains are transferred to the PC2 hood and washed three times with enough volume of cold sterile HEM to remove possible contaminants like blood and hair. The brains are then transferred to a 10cm Petri dish containing HEM.</li>
	<li>To dissect the forebrain periventricular region, the dish containing the brains is placed under the dissecting microscope with low magnification. The brains are then positioned flat on their ventral surface and held from the caudal side using fine curved forceps. Another set of curved forceps is used to remove the olfactory bulbs.</li>
	<li>After the removal of the olfactory bulbs, the brains are rotated to expose the ventral aspect.</li>
	<li>A 90 &deg; coronal cut is made at the level of the optic chiasm and the caudal aspects of the brains are discarded.</li>
	<li>This procedure is repeated until all brains are sectioned.</li>
	<li>At higher magnification, the rostral aspects of the brains are rotated so that the cut surface faces upward. First, the septum is removed and discarded using a fine curved micro-scissors or curved, pointed forceps, and then the thin layer of tissue surrounding the lateral wall of the ventricles is cut, excluding the striatal parenchyma and the corpus callosum. Dissected tissue is pooled in a 10cm sterile glass Petri dish.</li>
	<li>This procedure is repeated until all brains are micro-dissected.</li>
</ol>
Part 3: Tissue preparation and plating cells:
<ol>
	<li>The pooled tissue is minced for about 1-2 minutes using a scalpel blade, until only very small pieces remain.</li>
	<li>A total volume of 3 ml of %0.05 trypsin-EDTA is used and all of the minced tissue is transferred into a 15-ml tube. 3ml trypsin-EDTA is enough for a good digestion of tissues harvested from up to 8 mice.</li>
	<li>The tube is incubated for 7 min in a 37&deg;C water bath.</li>
	<li>At the end of the enzymatic incubation, the tube is returned to the hood and an equal volume of soybean trypsin inhibitor is added to stop trypsin activity.</li>
	<li>The suspension is pipetted up and down to ensure trypsin inactivation and then pelleted by centrifugation at 700 rpm (110 g) for 5min.</li>
	<li>The supernatant is discarded, and the tissue pieces are resuspended in 150 &mu;l of sterile NSC basal medium. Resetting the pipette to 200 &mu;l, the clumps are dissociated by gently pipetting up and down (3-7 times) until a smooth milky single cell suspension is achieved. The number of pippetting steps directly depends on the size of particles in the minced tissue (it is better to mince the tissue into very small pieces so as to increase the surface area for trypsin activity). Lengthy and vigorous mechanical dissociation might lead to reduced sphere formation due to neural stem and progenitor cell death.</li>
	<li>To remove debris and un-dissociated pieces, basal NSC medium is added to the dissociated cell suspension to reach a total volume of 10-15ml. The cell suspension is passed through a 40&mu;m cell strainer into an appropriate size tube, and then pelleted by centrifugation at 700 rpm (110 g) for 5min.</li>
	<li>To further remove the debris, it is recommended to resuspend the pellet in 10-15 ml of NSC medium and centrifuge the cell suspension at 700 rpm (110 g) for 5min.</li>
	<li>The supernatant is discarded. Then the cells are resuspended in appropriate volume of complete NSC medium supplemented with 20ng/ml EGF, 10ng/ml bFGF and 1&mu;l/ml of 0.2% heparin.</li>
	<li>Dissociated tissue from one brain is put into one T25 flask (containing 5 ml of complete medium). The cells are then incubated at 37&deg;C, 5% CO2 for 7-10 days by which time neurospheres should have formed. Tissue harvested from one brain usually can generate 400-600 neurospheres.</li>
</ol>
Part 4: Passaging and expansion of NSCs:
<ol>
	<li>When the neurospheres are ready for subculture (150-200 &mu;m in diameter), the medium with suspended spheres is removed from the flasks, placed in an appropriate size sterile tissue culture tube, and centrifuged at 700 rpm (110 g) for 5 min at room temperature.</li>
	<li>The supernatant is discarded and the spheres are resuspended in 1 ml of %0.05 trypsin-EDTA. Alternatively, neurospheres can also be passaged using mechanical or chemical dissociation methods described in the literature 2,3.</li>
	<li>The cell suspension is incubated at 37&deg;C in a water bath for 2-3 min, then an equal volume of soybean trypsin inhibitor is used to stop the trypsin activity.</li>
	<li>The cell suspension is gently pipetted up and down to ensure that the trypsin has been completely inactivated.</li>
	<li>The cell suspension is centrifuged at 700 rpm for 5 min. Then, the supernatant is removed and the cells are resuspended in 1 ml of 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.</li>
	<li>The cells are plated at a concentration of 5x104 cells/ml in complete NSC medium supplemented with 20ng/ml EGF, 10ng/ml bFGF and 1&mu;l/ml of 0.2% heparin in an appropriate size tissue culture flask. Use 5ml medium for T25, 20 ml for T75 and 40 ml for T175 flasks.</li>
	<li>Secondary neurospheres are formed in 5-7 days when incubated at 37&deg; C in a humidified incubator with 5% CO2.</li>
</ol>
Part 5: Representative Results:
In primary adult NSC culture, the majority of cells will die after 2-3 days but growth factor responsive precursor (including stem and progenitor cells) cells will proliferate (Figure 1) and generate colonies of undifferentiated cells after 6-8 days. In these cultures a significant amount of debris is normally present that might acquire a spherical shape, and can be mistaken for neurospheres. The amount of debris is directly dependent on the micro-dissection and tissue preparation techniques. True neurospheres are phase bright (luminous) and become more spherical as size increases (see video). After 7-10 days, the spheres must be rounded but not compacted; and should measure between 150 and 200 &mu;m and also should have rigid micro-spikes at the periphery at high magnification (Figure 2). If the neurospheres are allowed to grow too large, they become dark in color due to cell death at the center of the spheres. The large neurospheres are difficult to dissociate and eventually begin to attach to the substrate and differentiate.
<img src="/files/ftp_upload/2393/2393fig1.jpg" alt="Figure 1" /> Figure 1. Primary adult NSC culture 3 days after plating. Arrows show small colonies of proliferative NSCs. Original Magnification; 20x.
<img src="/files/ftp_upload/2393/2393fig2.jpg" alt="Figure 2" /> Figure 2. Passage one adult neurosphere 7 days after plating. Note the micro-spikes at the periphery of the sphere. Original Magnification; 20x.

<ol>
	<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><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>Sen, A., Kallos, M. S., Behie, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tissue+dissociation+protocol+for+scaled-up+production+of+neural+stem+cells+in+suspension+bioreactors.">New tissue dissociation protocol for scaled-up production of neural stem cells in suspension bioreactors.</a> Tissue Eng. 10, 904-913 (2004).</li><li>Morshead, C. M. <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+in+the+adult+mammalian+forebrain:+a+relatively+quiescent+subpopulation+of+subependymal+cells.">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> Neuron. 13, 1071-1082 (1994).</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>Dontu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes Dev. 17, 1253-1270 (2003).</li><li>Messina, E. <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+adult+cardiac+stem+cells+from+human+and+murine+heart.">Isolation and expansion of adult cardiac stem cells from human and murine heart.</a> Circ Res. 95, 911-921 (2004).</li><li>Ignatova, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cortical+glial+tumors+contain+neural+stem-like+cells+expressing+astroglial+and+neuronal+markers+in+vitro.">Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro.</a> Glia. 39, 193-206 (2002).</li><li>Galli, R. <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+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64, 7011-7021 (2004).</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></ol>

No conflicts of interest declared.

The neurosphere assay 2 has gained broad attention in the research community not only for the isolation and study of NSCs from CNS 4-5 but also for the isolation of other types of putative stem cells from numerous tissues, such as breast 6 and heart 7 and for the identification of brain, breast and colon tumor stem cells 8-9 suggesting that this culture system can be applied to a number of somatic and tumor precursor cell populations throughout the body. Some of the ...

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		<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>Stem Cell Technologies</td>
<td>05700</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>Stem Cell Technologies</td>
<td>05701</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>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>EGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>2028-EG</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<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>
<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>Fine curved forceps</td>
<td>Surgical tools</td>
<td>Fine Science Tools</td>
<td>11251&#x2010;35</td>
<td>&nbsp;</td>
<tr>
<td>Small fine forceps</td>
<td>Surgical tools</td>
<td>Fine Science Tools</td>
<td>11272&#x2010;30</td>
<td>&nbsp;</td>
<tr>
<td>Small forceps</td>
<td>Surgical tools</td>
<td>Fine Science Tools</td>
<td>11050&#x2010;10</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>		</tbody>
		</table>
		</div>
			<div>
			*To make HEM, mix 1&#215;10L packet of MEM and160ml 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.		</div>

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.

Watch this Scientific Journal Video about Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay at JoVE.com

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

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 ...

isolation-expansion-adult-mouse-neural-stem-cells-using-neurosphere

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

26.8K Views.  Shiraz University of Medical Sciences, Shiraz, Iran. 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. 

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

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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

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

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

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

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

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

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

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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 ...

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

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

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

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

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

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

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

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

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

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

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 ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

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

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Chapters in this video

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

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

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

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

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

Isolation and Culture of Embryonic Mouse Neural Stem Cells

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice