Name: one mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice
Uploaded: 2014-02-25T14:15:00
Duration: 9 min 52 s
Description: Watch this Scientific Journal Video about One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice at JoVE.com

TLW was supported by a Marie Curie International Incoming Fellowship. This work was also financed from basic institutional funding, Bundesministerium f&#252;r Bildung and Forschung (BMBF) funding and partly with support from Priority Research Program (SFB) 655 to GK. The authors would like to thank Anne Karasinsky for care and maintenance of all animals used in this study and Odette Leiter, Susann Ruhwald, Fanny Boehme, and Richard Wetzel for cell culture and microscopy assistance.

1. Basic Setup and Preparation of Culture Medium
<ol>
	<li>At least two days prior to commencing the experiment, prepare Poly-D-lysine (PDL)/Laminin coated plates for adherent monolayer cultures. To prepare wells/flasks add enough PDL (10 &#181;g/ml in dH2O) to coat the surface and incubate overnight at room temperature. Remove the solution from the dish and wash the dish three times with dH2O. Allow to air dry. Add Laminin (5 &#181;g/ml in cold DMEM:F12) and incubate at 37 &#176;C overnight. Remove the Laminin and either use the plates immediately or store with the Laminin at -20 &#176;C until required.</li>
	<li>Prepare fire polished pipettes with &#34;medium&#34; and &#34;small&#34; bores by rotating glass Pasteur pipettes in a flame until the edges become rounded. Autoclave to sterilize.</li>
	<li>On the day of dissection, prepare the appropriate amount of culture medium by mixing Neural Basal Medium with 2% B27, 1x GlutaMAX, 2 &#181;g/ml heparin, 50 units/ml Penicillin/Streptomycin, 20 ng/ml purified mouse receptor-grade epidermal growth factor (EGF), and 20 ng/ml recombinant bovine fibroblast growth factor (FGF-2). Warm the culture medium to 37 &#176;C in a water bath.</li>
	<li>For the SVZ dissociation, prepare 0.05% Trypsin-EDTA and 0.125 mg/ml Trypsin inhibitor containing 0.01 mg/ml DNaseI. Equilibrate these solutions to 37 &#176;C.</li>
	<li>Set up a dissection microscope and prepare the tools needed to remove the brain (scissors and small spatula) and for SVZ and DG dissections (scalpel, 27 G needle attached to 1 ml syringe, 1 x #7 forceps, 1 x #5/45 forceps) by soaking in 70% ethanol.</li>
</ol>
2. Harvesting of Adult Mouse Brains and SVZ/DG Microdissections
<ol>
	<li>Anesthetize single adult (8-week-old) mice according to the appropriate institutional guidelines. Perform cervical dislocation.</li>
	<li>Spray the head with 70% ethanol to sterilize the area and to minimize the amount of fur that adheres to the scissors and brain. Using sharp scissors decapitate the animal at the base of the brain stem.</li>
	<li>Holding the head at the base of the skull, cut the skull between the two olfactory bulbs by placing one blade of a small pair of scissors into each eye cavity and cutting coronally. Next, make two lateral cuts at the base of the skull, followed by a longitudinal cut through the skull along the sagittal suture. Caution: ensure the angle of the scissors is as shallow as possible to avoid damaging the underlying brain.</li>
	<li>Expose the brain by peeling back the skull with either the blade of the scissors or a pair of curved forceps. Free the brain from the skull using a small spatula and place into cold PBS.</li>
	<li>Rinse brains with PBS to remove blood and fur.</li>
	<li>Transfer brains to a 10 cm plastic Petri dish containing PBS</li>
	<li>Place Petri dish containing the brain under a dissecting microscope at low magnification and position the brain on its ventral surface. Using fine curved forceps remove the olfactory bulbs while holding the brain in position by the cerebellum.</li>
	<li>Rotate the brain onto the dorsal aspect and using a scalpel make a coronal cut through the brain at the level of the optic chiasm</li>
	<li>To microdissect the SVZ (for more detailed instructions, see also Azari&#160;et al.10), place the rostral portion of the brain so that the cut coronal surface faces upwards and focus the microscope onto a higher magnification. Remove and discard the septum using fine curved forceps.</li>
	<li>Dissect the SVZ (the thin layer of tissue surrounding the ventricle) by placing the tip of one blade of a pair of fine curved forceps in the lateral corner of the lateral ventricle immediately under the corpus callosum and the other approximately 1 mm into the tissue immediately adjacent to the ventricle. Press down the forceps towards the base of the dish and towards the ventral aspect of the ventricle to remove a small triangular piece of tissue. Place the dissected SVZ into a Petri dish on ice.</li>
	<li>To microdissect the DG (for more detailed instructions, see also Hagihara&#160;et al.11), place the caudal portion of the brain in the Petri dish and cut along the longitudinal fissure using a scalpel.</li>
	<li>Under a dissection microscope, remove the cerebellum and the diencephalon using forceps.</li>
	<li>Refocus the microscope so that the borders around the DG are now visible. To remove the dentate gyrus, insert the tip of a 27 G&#160;needle and slide along the border between the DG and Ammon&#39;s horn. Using the fine forceps, free the DG from the surrounding tissue.</li>
</ol>
3. SVZ Tissue Dissociation
<ol>
	<li>Mince the tissue using a scalpel blade for approximately 1 min until no large pieces remain.</li>
	<li>Transfer the minced tissue to a 15 ml tube using 1 ml of prewarmed 0.05% Trypsin-EDTA and incubate for 7 min in a water bath set to 37 &#176;C.</li>
	<li>To stop the enzymatic reaction, add 1 ml of trypsin inhibitor containing DNaseI and mix the contents by flicking the tube.</li>
	<li>Pellet the suspension by centrifugation at 300 x g for 5 min and discard the supernatant</li>
	<li>Resuspend the pellet in 1 ml of growth medium and dissociate by gently pipetting up and down approximately 7-10x using a P1000 pipette. Caution: over triturating can lead to increased cell death and will negatively impact on subsequent cell growth.</li>
	<li>Add growth medium to a total volume of 5 ml and pass the cell suspension through a 40 &#181;m sieve to remove debris and undissociated tissue clumps.</li>
	<li>Centrifuge at 300 x g for 5 min, discard the supernatant and resuspend the resulting pellet in 200 &#181;l growth medium.</li>
</ol>
4. DG Tissue Dissociation
<ol>
	<li>Mince the tissue using a scalpel blade for approximately 1 min until no large pieces remain and transfer into prewarmed PDD enzyme mix (Papain 2.5 U/ml, Dispase 1 U/ml, DNaseI 250 U/ml). Incubate for 20 min at 37 &#176;C, mixing by inverting the tube every 3-5 min.</li>
	<li>Dissociate the tissue mechanically using a medium bore, fire polished, Pasteur pipette by pipetting up and down gently 10x.</li>
	<li>Incubate for a further 10 min at 37 &#176;C, mixing by inverting the tube every 3-5 min.</li>
	<li>Further dissociate the tissue mechanically using a small bore, fire polished Pasteur pipette by pipetting up and down gently 10x.</li>
	<li>Centrifuge at 130 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 1 ml buffer solution (1x HBSS, 30 mM Glucose, 2 mM HEPES (pH 7.4), 26 mM NaHCO3). Make up to 10 ml with buffer solution.</li>
	<li>Centrifuge at 130 x g for 5 min.</li>
	<li>Remove supernatant and resuspend the pellet in 5 ml of 20% Percoll. (To prepare 90% Percoll, add 4.5 ml of 100% Percoll to 0.5 ml of 10x&#160;PBS then further dilute this to 20% by adding 1.1 ml 90% Percoll to 3.9 ml 1x&#160;PBS).</li>
	<li>Centrifuge 450 x g for 15 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 10 ml buffer.</li>
	<li>Centrifuge at 130 x g for 5 min.</li>
	<li>Resuspend the pellet in 200 &#181;l growth medium.</li>
</ol>
5. Generation of Adherent Monolayer Cultures
<ol>
	<li>Plate the dissociated SVZ or DG tissue into a single PDL/Laminin coated well of a 96-well plate and incubate at 37 &#176;C with 5% CO2.</li>
	<li>Approximately 24 hr after plating, once the cells have adhered to the coated surface, exchange the growth medium to further remove excess debris.</li>
	<li>Every subsequent 3-4 days, exchange half of the growth medium with fresh medium to replenish the growth factors.</li>
	<li>Repeat until the cells reach approximately 80% confluency and are ready to be passaged. Note: the time between plating and the first passage can take up to 2-3 weeks.</li>
</ol>
6. Passaging of Adherent Monolayer Cultures
<ol>
	<li>When the cultures reach approximately 80% confluency remove the medium from the well and wash with PBS. 
	Note: Do not allow the cells to exceed 90% confluency as this can lead to detachment of cells and neurosphere formation and in addition, increased levels of cell death.</li>
	<li>Add 50 &#181;l Accutase and incubate at 37 &#176;C for 2-3 min (checking to see if the cells are rounded and detached).</li>
	<li>Remove the cells to a 15 ml tube and wash the well once with PBS and transfer to the same tube.</li>
	<li>Dilute cells to 5 ml with PBS and centrifuge 300 x g for 5 min.</li>
	<li>For the first passage, dilute cells to 1 ml and plate into a PDL/Laminin coated well of a 24 well plate.</li>
	<li>For subsequent passages, resuspend cells in 200 &#181;l growth medium and count using a hemocytometer. Plate at 1 x 104 cells/cm2 in the appropriate sized coated well or flask.</li>
</ol>
7. Differentiation of Adherent Monolayer Cultures
<ol>
	<li>To differentiate the adherent monolayer cultures, plate proliferating cells onto PDL/Laminin coated coverslips at a density of 1 x 104 cells/cm2 in growth medium containing 20 ng/ml EGF and 10 ng/ml bFGF.</li>
	<li>When the cells reach approximately 80% confluency (usually 2 days), replace the growth medium with medium containing 5 ng/ml bFGF and 0 ng/ml EGF.</li>
	<li>Following 2 days in 5 ng/ml bFGF, replace the medium with growth medium in the absence of both mitogens for a further 3 days. Note: during this period a significant amount of cell death will occur.</li>
	<li>After a total of 5 days, wash the differentiated cells with PBS to remove any dead cells then fix with 4% paraformaldehyde (PFA) for 20 min at room temperature.</li>
	<li>Wash again with PBS to remove any PFA and store coverslips in wells in 1 ml PBS at 4 &#176;C.</li>
</ol>
8. Neurosphere Culture and Quantification
<ol>
	<li>Dilute the dissociated SVZ or DG tissue from one animal in 20 ml of culture medium and plate 200 &#181;l/well across a 96-well plate using a 10 ml multidoser pipette.</li>
	<li>Incubate at 37 &#176;C with 5% CO2 for 6-7 days for SVZ-derived neurospheres and 10-12 days for DG-derived neurospheres. 
	Note: growth for longer than these recommended incubation times will result in overgrowth and will lead to cell death in the center of the neurospheres and/or, spontaneous attachment and differentiation.</li>
	<li>Count and measure the diameter of the neurospheres using an eyepiece graticule fitted to an upright light microscope</li>
</ol>
9. Passaging the Neurospheres
After the primary neurospheres have been counted and their size recorded they can be expanded over several passages beginning with either a single neurosphere or a bulk culture.
<ol>
	<li>Bulk culture neurosphere expansion
	<ol>
		<li>To passage combined neurospheres as a bulk culture, remove the medium containing the neurospheres from the plate, transfer to a 15 ml tube and centrifuge at 300 x g for 5 min.</li>
		<li>Discard the supernatant and resuspend the neurospheres in 1 ml of prewarmed 0.05% Trypsin-EDTA and incubate at room temperature for 3 min.</li>
		<li>Add an equal volume of trypsin inhibitor containing DNaseI and mix well.</li>
		<li>Centrifuge for 5 min at 300 x g, remove the supernatant and add 1 ml of growth medium.</li>
		<li>Triturate up and down approximately 10x with a P1000 pipette to dissociate the neurospheres.</li>
		<li>Remove 10 &#181;l of the cell suspension and mix with an equal volume of trypan blue and perform a live cell count using a hemocytometer.</li>
		<li>Reseed the cells at a density of 1 x 104 cells/cm2 in the appropriate sized cell culture well or flask.</li>
		<li>Incubate at 37 &#176;C with 5% CO2 until secondary neurospheres form.</li>
	</ol>
	</li>
	<li>Single neurosphere expansion
	<ol>
		<li>To passage individual neurospheres choose wells that contain a single neurosphere and carefully remove and discard approximately 160 &#181;l of the growth medium from each well without disturbing the neurosphere.</li>
		<li>Add 100 &#181;l of 0.05% Trypsin-EDTA to each well to be passaged and incubate at room temperature for 3 min.</li>
		<li>Add 100 &#181;l of Trypsin inhibitor containing DNAseI to stop the reaction.</li>
		<li>Triturate approximately 10 times up and down with a P200 pipette to break apart the neurosphere.</li>
		<li>Transfer the 200 &#181;l containing the dissociated neurosphere to a new well of a 24-well plate containing 1.5 ml of growth medium. Incubate at 37 &#176;C with 5% CO2 until secondary neurospheres form. 
		Note: to determine long-term potential, one of the cardinal properties of a true neural stem cell, neurospheres should be passaged for at least 5-10 passages. See also the literature on the in part controversial interpretation of such results12-14.</li>
	</ol>
	</li>
</ol>
10. Differentiation of Neurosphere Cultures
Primary or passaged neurospheres can be differentiated to determine multipotentiality.
<ol>
	<li>Remove neurospheres in suspension from their culture plate or flask and transfer them to a 10 cm plastic Petri dish.</li>
	<li>Under a dissection microscope remove approximately 15-20 neurospheres from the medium using a P20 pipette and transfer to a 24-well plate containing the culture medium without growth factors and a PDL/Laminin coated coverslip.</li>
	<li>Differentiate for approximately 7 days at 37 &#176;C with 5% CO2.</li>
	<li>Wash the differentiated neurospheres with PBS to remove any dead cells then fix with 4% PFA for 20 min at room temperature.</li>
	<li>Wash again with PBS to remove any PFA and store coverslips in wells in 1 ml PBS at 4 &#176;C</li>
</ol>
11. Immunostaining of Neurosphere and Adherent Cultures
Note: For staining with the O4 antibody omit the Triton from the blocking and staining steps and remember to use an appropriate IgM secondary antibody.
<ol>
	<li>Incubate the coverslips containing the differentiated neurospheres or adherent monolayer cultures in blocking solution (10% Normal Donkey Serum in PBS containing 0.2% Triton X-100) for 60 min at room temperature.</li>
	<li>Incubate in fresh blocking solution containing primary bIII-tubulin, Map2a+b, glial fibrillary acidic protein (GFAP) or O4 antibodies for 60min at room temperature.</li>
	<li>Wash 3x with PBS.</li>
	<li>Incubate in fresh blocking solution containing appropriate fluorescence conjugated secondary antibodies and 4,6-diamidino-2-phenylindole (DAPI; 1:5,000) for 30 min at room temperature in the dark.</li>
	<li>Wash three times with PBS.</li>
	<li>Mount the coverslips onto microscope slides using fluorescence mounting medium and air dry in the dark overnight</li>
	<li>View and image using a fluorescence microscope.</li>
</ol>

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The authors have nothing to disclose.

This paper presents a detailed protocol for the initiation of neural precursor cultures, both as adherent monolayers and neurospheres, from the two major neurogenic regions of the adult mouse brain. There are a number of important points that must be kept in mind when attempting either of these in vitro culture systems. Firstly, the choice of dissociation method is very important and is tissue dependent. In our hands, 0.05% trypsin-EDTA is very effective for dissociation of SVZ tissue, and results in a higher number of neurospheres than when using a papain-based dissociation technique. For the dissociation of DG tissue however, we strongly recommend a papain-based dissociation approach. When directly comparing the two dissociation methods on DG tissue, we observed a significantly lower yield of viable cells and approximately 10-fold fewer neurospheres when using trypsin. This difference in dissociation could be due to the difference in tissue composition between the two regions. The compact tissue of the DG is surrounded by extensive neuropil and extensive damage of cellular processes can occur during dissociation.
A second important point to note is that, while the neurosphere assay can be useful to make quantitative statements about the number of precursor cells present in a given tissue sample, some caution must, however, be employed in the interpretation of these absolute numbers. Fusion of neurospheres can be a major confounding factor. Several studies have shown that neurons are highly motile and can fuse, even under what are supposedly &#8216;clonal&#8217; conditions7,19. The resulting neurosphere frequency can be very dependent on factors including the medium components, the dissection procedure and the dissociation process. Even between experienced handlers some variation in the number of neurospheres generated from supposedly identical samples is evident (see Figure 1A.) More useful, is a direct comparison of the precursor frequency between two given samples (i.e. control vs. treated or wild-type vs. knock-out) handled by the same person within a single experiment, rather than a quantitative statement of total precursor cell number.
When deciding which of the two culture methods is most suited for a particular experiment it is important to note that these two culture systems differ in the homogeneity of the cell types generated. In comparison to proliferating adherent cell cultures, which show a fairly homogeneous precursor cell pool (~98% of cells are Sox2+)20, neurospheres are more heterogeneous and contain, as well as proliferating precursor cells, differentiated neurons, and astrocytes21,22. It is important that the neurospheres are not cultured for extended periods between passages as the larger the neurosphere become the more likely it is to find differentiated cell types in their core.
We traditionally initiate adherent monolayer neural precursor cultures from the DG tissue of between 5-8 mice. Therefore, when attempting to establish the adherent monolayer cultures from the DG or SVZ of a single mouse, the utmost care needs to be taken during the tissue dissociation procedure in order to avoid excessive cell death caused by over triturating of the tissue, or taking extended periods of time between the dissection and final culturing steps. This protocol describes, for the first time, the generation of adherent monolayer precursor cultures from both the SVZ and DG of individual animals. There are many instances when the comparison of precursor proliferation and differentiation needs to be made on a single animal basis. These include the ability to directly compare the DG and SVZ of individual animals using paired statistics and to pair culture data with individual behavioral or physiological data9. Single animal cultures also allow the use of rare transgenic animals, where age-matching a pool of 5-8 donors per culture is not possible, as well as unique animals (e.g. F2 crosses or out bred animals) for genetic association studies.

A correction was made to <a href="/video/51225">One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice</a>. Many micro symbols were changed into milli symbols by accident: In the Protocols, sections 1.1, 1.3, 3.6, 3.7, 6.2, 6.6, 8.1, 9.1.6, 9.2.2, and 9.2.5 need to be fixed, as does Figure 1 description in the Results section.
Protocol section 1.1 was changed from:
At least two days prior to commencing the experiment, prepare Poly-D-lysine (PDL)/Laminin coated plates for adherent monolayer cultures. To prepare wells/flasks add enough PDL (10 mg/ml in dH2O) to coat the surface and incubate overnight at room temperature. Remove the solution from the dish and wash the dish three times with dH2O. Allow to air dry. Add Laminin (5 mg/ml in cold DMEM:F12) and incubate at 37 &#176;C overnight. Remove the Laminin and either use the plates immediately or store with the Laminin at -20 &#176;C until required.
to:
At least two days prior to commencing the experiment, prepare Poly-D-lysine (PDL)/Laminin coated plates for adherent monolayer cultures. To prepare wells/flasks add enough PDL (10 &#181;g/ml in dH2O) to coat the surface and incubate overnight at room temperature. Remove the solution from the dish and wash the dish three times with dH2O. Allow to air dry. Add Laminin (5 &#181;g/ml in cold DMEM:F12) and incubate at 37 &#176;C overnight. Remove the Laminin and either use the plates immediately or store with the Laminin at -20 &#176;C until required.
Protocol section 1.3 was changed from:
On the day of dissection, prepare the appropriate amount of culture medium by mixing Neural Basal Medium with 2% B27, 1x GlutaMAX, 2 &#181;g/ml heparin, 50 units/ml Penicillin/Streptomycin, 20 ng/ml purified mouse receptor-grade epidermal growth factor (EGF), and 20 ng/ml recombinant bovine fibroblast growth factor (FGF-2). Warm the culture medium to 37 &#176;C in a water bath.
to:
On the day of dissection, prepare the appropriate amount of culture medium by mixing Neural Basal Medium with 2% B27, 1x GlutaMAX, 2 mg/ml heparin, 50 units/ml Penicillin/Streptomycin, 20 ng/ml purified mouse receptor-grade epidermal growth factor (EGF), and 20 ng/ml recombinant bovine fibroblast growth factor (FGF-2). Warm the culture medium to 37 &#176;C in a water bath.
Protocol section 3.6 was changed from:
Add growth medium to a total volume of 5 ml and pass the cell suspension through a 40 mm sieve to remove debris and undissociated tissue clumps.
to:
Add growth medium to a total volume of 5 ml and pass the cell suspension through a 40 &#181;m sieve to remove debris and undissociated tissue clumps.
Protocol section 3.7 was changed from:
Centrifuge at 300 x g for 5 min, discard the supernatant and resuspend the resulting pellet in 200 ml growth medium.
to:
Centrifuge at 300 x g for 5 min, discard the supernatant and resuspend the resulting pellet in 200 &#181;l growth medium.
Protocol section 6.2 was changed from:
Add 50 ml Accutase and incubate at 37 &#176;C for 2-3 min (checking to see if the cells are rounded and detached).
to:
Add 50 &#181;l Accutase and incubate at 37 &#176;C for 2-3 min (checking to see if the cells are rounded and detached).
Protocol section 6.6 was changed from:
For subsequent passages, resuspend cells in 200 ml growth medium and count using a hemocytometer. Plate at 1 x 104 cells/cm2 in the appropriate sized coated well or flask.
to:
For subsequent passages, resuspend cells in 200 &#181;l growth medium and count using a hemocytometer. Plate at 1 x 104 cells/cm2 in the appropriate sized coated well or flask.
Protocol section 8.1 was changed from:
Dilute the dissociated SVZ or DG tissue from one animal in 20 ml of culture medium and plate 200 ml/well across a 96-well plate using a 10 ml multidoser pipette.
to:
Dilute the dissociated SVZ or DG tissue from one animal in 20 ml of culture medium and plate 200 &#181;l/well across a 96-well plate using a 10 ml multidoser pipette.
Protocol section 9.1.6 was changed from:
Remove 10 ml of the cell suspension and mix with an equal volume of trypan blue and perform a live cell count using a hemocytometer.
to:
Remove 10 &#181;l of the cell suspension and mix with an equal volume of trypan blue and perform a live cell count using a hemocytometer.
Protocol section 9.2.2 was changed from:
Add 100 ml of 0.05% Trypsin-EDTA to each well to be passaged and incubate at room temperature for 3 min.
to:
Add 100 &#181;l of 0.05% Trypsin-EDTA to each well to be passaged and incubate at room temperature for 3 min.
Protocol section 9.2.5 was changed from:
Transfer the 200 ml containing the dissociated neurosphere to a new well of a 24-well plate containing 1.5 ml of growth medium. Incubate at 37 &#176;C with 5% CO2 until secondary neurospheres form.
to:
Transfer the 200 &#181;l containing the dissociated neurosphere to a new well of a 24-well plate containing 1.5 ml of growth medium. Incubate at 37 &#176;C with 5% CO2 until secondary neurospheres form.
Figure 1 description was updated from:
Figure 1. Adult mouse precursor cells can be cultured as adherent monolayer cultures (A) or as neurospheres (B: SVZ, C: DG). Scale bar is 50 mm.
to:
Figure 1. Adult mouse precursor cells can be cultured as adherent monolayer cultures (A) or as neurospheres (B: SVZ, C: DG). Scale bar is 50 &#181;m.

A correction was made to <a href="/video/51225">One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice</a>. Many micro symbols were changed into milli symbols by accident: In Protocols, sections 1.1, 1.3, 3.6, 3.7, 6.2, 6.6, 8.1, 9.1.6, 9.2.2, and 9.2.5 need to be fixed, as does the Figure 1 description in Results.

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

The neurosphere assay1,2 and the adherent monolayer culture3,4, both developed in the early 1990s, still remain the gold standard in vitro neural stem cell assays. In these assays, primary tissue is micro dissected from a particular brain region, dissociated into a single cell suspension and cultured in the presence of the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) to form either free-floating clusters (neurospheres) or adherent monolayers. Both systems have a number of advantages and disadvantages and careful consideration should be given to the question that is to be addressed before one or the other system is chosen.
Neurospheres allow a straightforward read-out of differences in precursor cell number and potential. In addition, neurospheres are also a useful tool to study intrinsic specification of the cells when removed from their normal external environment. Extrinsic cues can be studied by simply adding the factor of interest to the growth medium and quantifying the number and size of the neurospheres generated. The major drawback of neurospheres however, is that they form their own niche, with the cells at the center of the neurospheres (particularly large neurospheres) being more differentiated than those on the surface5. Neurospheres contain a mix of stem cells, committed progenitors, and differentiated cells and the cell-cell interactions within the neurospheres counteract the maintenance of the stem cells. This is why neurospheres contain only a small number of true stem cells6-8.
Adherent monolayer cultures also provide a good in vitro system to model in vivo proliferation. Adherent cultures, in which the cells remain more isolated and homogeneous, can eliminate the heterogeneous nature of the neurosphere. Under these growth conditions the precursor cells proliferate rapidly and almost all cells are dividing and express the characteristic neural precursor markers Nestin, Sox2, and BLBP. The major disadvantage of the monolayer culture system compared to the neurosphere assay is that individual precursor-derived clones are unable to be monitored and quantified.
A drawback of most protocols for both types of cultures has been the necessity to use relatively large numbers of animals, because the yield of the isolation strategies has often been poor. At the same time, it has become clear that adult neurogenesis contributes to the individualization of the brain9, resulting in the need for individualized ex vivo models as well. These needs can be met by &#34;one-mouse-one-culture&#34; protocols as described in this report.
The following visual protocol describes the simultaneous generation of neural precursor cultures from both the SVZ and DG of individual animals either as adherent monolayers or as neurospheres. The generation of cultures from individual animals is particularly useful when comparisons between individually treated animals or various individual transgenic or wild-type mice are required. This protocol includes detailed instructions for the simultaneous microdissection of the SVZ and DG regions from adult mice, their dissociation into a single cell suspension, in vitro culture as either adherent monolayer cultures or neurospheres and analysis of multipotentiality and long-term potential, the two cardinal properties of a bone fide stem cell.

<table border="0" cellpadding="0" cellspacing="0" width="763">
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	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">CONSUMABLES</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">poly-D-lysine</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">P7280-5MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">laminin</td>
			<td style="width:179px;">Roche</td>
			<td style="width:119px;">11243217001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">glass pastuer pipettes</td>
			<td style="width:179px;">Volac</td>
			<td style="width:119px;">BS5732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">DMEM:F12 (1:1) 1X</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:119px;">21331-020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Neural Basal Medium (1X)</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">B27 supplements (50X)</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Glutamax</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:119px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Heparain</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">H3393</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Penacillin/Streptomycin</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">EGF</td>
			<td style="width:179px;">PeproTech</td>
			<td style="width:119px;">AF-100-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">bFGF</td>
			<td style="width:179px;">PeproTech</td>
			<td style="width:119px;">100-18B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">0.05% trypsin-EDTA</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">trypsin inhibitor</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">T6522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">DNaseI</td>
			<td style="width:179px;">Roche</td>
			<td style="width:119px;">10104159001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Accutase</td>
			<td style="width:179px;">PAA</td>
			<td style="width:119px;">L11-007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Papain</td>
			<td style="width:179px;">Worthington</td>
			<td>LS003120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Dispase</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:119px;">17105-041</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Percoll</td>
			<td style="width:179px;">GE Healthcare</td>
			<td style="width:119px;">17-0891-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">HBSS (with Calcium &#38; Magnesium)</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:119px;">14025-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Glucose</td>
			<td style="width:179px;">Roth</td>
			<td style="width:119px;">X997.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">HEPES</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">H3375-500G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:299px;">NaHCO3</td>
			<td style="width:179px;">Merck</td>
			<td style="width:119px;">K39347429847</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">1mL syringes&#160;</td>
			<td style="width:179px;">Braun</td>
			<td style="width:119px;">2016-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">27 Gauge needles</td>
			<td style="width:179px;">Braun</td>
			<td style="width:119px;">4657705</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">scalpels (#22 disposable)</td>
			<td style="width:179px;">Braun</td>
			<td style="width:119px;">BA222</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Dumont #7 forceps</td>
			<td style="width:179px;">FST&#160;</td>
			<td style="width:119px;">11271-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Dumont 5/45 forceps</td>
			<td style="width:179px;">FST&#160;</td>
			<td style="width:119px;">11251-35</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">scissors</td>
			<td style="width:179px;">FST&#160;</td>
			<td style="width:119px;">14060-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Iris spatula</td>
			<td style="width:179px;">FST&#160;</td>
			<td style="width:119px;">10093-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">70% ethanol</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">PBS</td>
			<td style="width:179px;">Life Technologies</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">flasks/well plates</td>
			<td style="width:179px;">TPP</td>
			<td style="width:119px;">92696</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">PFA (4%)</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">hemocytometer</td>
			<td style="width:179px;">Marienfeld</td>
			<td style="width:119px;">650010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">trypan blue (0.4%)</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">NDS</td>
			<td style="width:179px;">Millipore</td>
			<td style="width:119px;">530</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">TritonX-100</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mouse monoclonal bIII-tubulin antibody</td>
			<td style="width:179px;">Promega</td>
			<td style="width:119px;">G712A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit polyclonal glial fibrillary acidic protein antibody</td>
			<td style="width:179px;">Dako</td>
			<td style="width:119px;">20334</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">O4&#160;</td>
			<td style="width:179px;">R &#38; D systems</td>
			<td style="width:119px;">MAB1326</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Map2a+b</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">M1406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:299px;">donkey anti-mouse Cy3 antibody&#160;</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">715-505-151</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey anti-rabbit Alexa488&#160;</td>
			<td style="width:179px;">Dianova</td>
			<td style="width:119px;">711-545-152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4,6-diamidino-2-phenylindole (DAPI)&#160;</td>
			<td style="width:179px;">Invitrogen</td>
			<td style="width:119px;">861405</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Aqua Polymount</td>
			<td style="width:179px;">Polysciences Inc</td>
			<td style="width:119px;">18606</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">10 ml Combi tips</td>
			<td style="width:179px;">eppendorf</td>
			<td style="width:119px;">30089677</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:299px;">plastic 10ml and 25mL serological pipettes</td>
			<td style="width:179px;">Corning</td>
			<td style="width:119px;">4488/4489</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">EQUIPMENT</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Pipetboy</td>
			<td style="width:179px;">Integra biosciences</td>
			<td style="width:119px;">521942</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">multidoser pipette</td>
			<td style="width:179px;">eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">37C waterbath</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">dissecting microscope</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">37C:5%Co2 incubator</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">centrifuge</td>
			<td style="width:179px;">eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Although the two neurogenic regions of the adult mouse brain both contain neural precursor cells, these cells can behave quite differently when cultured in vitro. The adherent monolayer cultures generated from both regions appear morphologically indistinguishable (Figure 1A), however, the SVZ-derived adherent cultures proliferate faster and need to be passaged, on average, 1-2 days earlier than those derived from the DG. As neurospheres, the SVZ-derived precursor cells also proliferate faster and form larger neurospheres (Figure 1B) than the DG-derived precursor cells (Figure 1C). Whilst SVZ-derived neurospheres are typically counted after 6-7 days in culture, DG-derived neurospheres are usually quantified after 10-12 days. In addition, a far greater number of neural precursor cells reside in the SVZ compared to the DG, as evidenced by the almost 10-fold greater number of neurospheres that can be generated from this region (SVZ: 1,173&#177;74.9 vs. DG: 145.3&#177;26.4; p = &#60;0.0001; n = 10 animals per group; Figure 2A).
Studies have shown that the precursor cells within the SVZ and DG respond to different stimuli. The precursor cells in the DG are activated by specific types of spatial learning and by stimuli such as environmental enrichment and physical activity, whereas the SVZ precursor cells are activated by olfactory learning and olfactory enrichment. Consistent with this, one of us (TLW) previously demonstrated that the DG contains a population of latent stem and progenitor cells that can be activated by neural excitation15-18. In contrast, we found that the SVZ precursor cells respond quite differently to this stimulus, with a decrease in neurosphere number in response to depolarizing levels of KCl 17. Here, we have repeated this experiment, plating half of the isolated cells derived from the SVZ and DG of individual animals in depolarizing levels of KCl and the other half in control KCl levels. We demonstrate, as previously, that while the DG precursor cells are activated by depolarization (101.2&#177;17.4 vs. 184.8&#177;12.5, p = 0.005, n = 5 animals), the proliferation of the SVZ-derived cells is in fact significantly decreased (368.0&#177;62.9 vs 266.6&#177;41.6, p = 0.02, n = 5 animals; Figure 2B).
To confirm long-term potential, one of the cardinal features of a true stem cell, single neurospheres or adherent monolayer cultures must be capable of extended expansion i.e. over at least 10 passages. At each passage, following the preparation of a single-cell suspension, the number of cells is counted and the fold expansion is calculated. The theoretical cell total is then calculated by multiplying the fold expansion during that passage by the theoretical total from the previous passage. This is displayed as a line graph with the passage number plotted against the log10 of the theoretical total cell number (see example Figure 3). To confirm multipotentiality, both monolayer cultures and neurospheres can be differentiated by mitogen withdrawal and be shown to give rise to both neurons, and glia (Figure 4).
<img alt="Figure 1" fo:content-width="3in" fo:src="/files/ftp_upload/51225/51225fig1highres.jpg" src="/files/ftp_upload/51225/51225fig1.jpg" /> 
Figure 1. Adult mouse precursor cells can be cultured as adherent monolayer cultures (A) or as neurospheres (B: SVZ, C: DG). Scale bar is 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/51225/51225fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51225/51225fig2highres.jpg" src="/files/ftp_upload/51225/51225fig2.jpg" /> 
Figure 2. Significantly more neurospheres are generated from the SVZ compared to the DG of single mice (A). The SVZ and DG precursor cells respond differently to in vitro depolarization (B).
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51225/51225fig3highres.jpg" src="/files/ftp_upload/51225/51225fig3.jpg" /> 
Figure 3. To confirm long-term potentiation, neurospheres are expanded for over 10 passages.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51225/51225fig4highres.jpg" src="/files/ftp_upload/51225/51225fig4.jpg" /> 
Figure 4. Neurospheres can be differentiated into bIII-tubulin+ neurons (A: red), GFAP+ astrocytes (A: green), O4+ oligodendrocytes (B: red) and Map2ab+ neurons (C: red). <a href="https://www.jove.com/files/ftp_upload/51225/51225fig4highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice at JoVE.com

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

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 ...

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Research

JoVE Journal

Neuroscience

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 ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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 ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

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 Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

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

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

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

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