Name: isolation and expansion of adult canine hippocampal neural precursors
Uploaded: 2016-11-29T15:00:00.000+00:00
Duration: 9 min 37 s
Description: Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia grants (#568969 and 1004152).

In accordance with New South Wales, Australia law, post mortem brain tissue was acquired from adult dogs euthanized for reasons unrelated to the study.
1. Preparation of Culture Medium
<ol>
	<li>Prepare a 0.1% gelatin solution by adding 0.1 g of gelatin powder to 100 ml of distilled water and agitating at 37 &#176;C until dissolved. Sterilize the solution by UV irradiation for 15 min. This media can then be stored at 4 &#176;C for up to 1 month.</li>
	<li>Prepare a 3:1 ratio of Dulbecco&#39;s Modified Eagle Medium (DMEM) and nutrient mixture F12 by adding 167 ml of F-12 to 500 ml of DMEM (4.5 g/L D-glucose). Add 6.7 ml of Penicillin/Streptomycin solution (for a 1% final concentration). This media can be stored in the dark at 4 &#176;C for up to 1 month.</li>
	<li>Prepare 500 ml of serum media by combining 440 ml of DMEM (4.5 g/L D-glucose), 5 ml of L-alanyl-L-glutamine dipeptide (200 mM), 5 ml of Penicillin/Streptomycin and 50 ml of fetal bovine serum (FBS). This media can be stored in the dark at 4 &#176;C for up to 1 month.</li>
	<li>Complete growth medium consists of Neural Stem Cell (NSC) Basal Medium and Proliferation Neural Supplement-A (NS-A), 2% FBS, 2 &#181;g/ml heparin, 20 ng/ml epidermal growth factor (EGF) and 10 ng/ml basic fibroblast growth factor (bFGF). Make this media fresh, immediately prior to brain dissection, and warm to 37 &#176;C in a water bath.</li>
</ol>
2. Brain Extraction
<ol>
	<li>Begin extraction within 6 hr post mortem by sterilizing the dorsal surface of the dog&#39;s head using povidone-iodine.</li>
	<li>Using a scalpel, make a longitudinal incision along the sagittal midline, across the entire dorsal surface of the cranium, to expose the underlying masseter muscles. The length of this cut will depend on the species and age of the dog.</li>
	<li>At the rostral and caudal borders of the cranium, make additional 5 - 10 cm perpendicular cuts on both sides, passing behind the eyes and ears respectively, to allow the skin to be fully reflected.</li>
	<li>Using a scalpel, separate the masseter muscles from their attachment to the sagittal crest of the skull and scrape any remaining connective tissue from the cranium.</li>
	<li>Using an oscillating bone saw cut the cap of the skull in a circumferential line. Using a probe or scalpel sever any remaining attachments with the dura, and then carefully remove the skull cap to expose the dorsal surface of the brain. 
	Caution: The thickness of the bone varies at different regions in the skull, so caution must be exercised to not penetrate too deep and damage the underlying brain.</li>
	<li>Sever the spinal cord by inserting a scalpel between the upper cervical vertebrae.</li>
	<li>With one hand to gently lift the brain, use a scalpel to carefully free the brain from the cranial fossae and sever the connecting nerves and blood vessels located on its ventral side. Gently lift the brain out of the skull and into a container of DPBS. 
	Caution: The large olfactory bulbs of the canine brain may extend deep into the anterior cranial fossa. Care must be taken when removing the brain so as to preserve these structures.</li>
</ol>
3. Hippocampal Dissection
<ol>
	<li>Rinse the brain in Dulbecco&#39;s phosphate buffered saline (DPBS) to remove any blood and place it dorsal side down onto a 150 mm Petri dish.</li>
	<li>Slowly bisect the brain longitudinally through the mid-sagittal plane using a wet brain knife and a single slice that utilizes the full length of the blade. Do not apply additional downward pressure, or use a sawing motion, as this may damage the tissue.</li>
	<li>Placing each hemisphere medial surface up, dissect out the hippocampus using a scalpel and fine forceps, and transfer it to a 35 mm tissue culture dish.</li>
</ol>
4. Isolation of Neural Precursor Cells
<ol>
	<li>Mince the tissue samples using a scalpel blade into approximately 1 mm3 pieces.</li>
	<li>Transfer the tissue into a 15 ml tube, add 2 ml of 0.1% Trypsin EDTA, and incubate for 7 min in a water bath at 37 &#176;C.</li>
	<li>Halt the enzyme reaction by adding 4 ml of serum media to the tube. Pellet the suspension by centrifugation at 100 x g for 7 min and remove the supernatant.</li>
	<li>Resuspended the pellet in 300 &#181;l of DPBS, and mechanically dissociated by gently pipetting up and down, using a 1,000 &#181;l pipette tip, until a smooth homogenate forms.</li>
	<li>Add 14 ml of serum media to the tube and pass the cell suspension through a 40 &#181;m cell strainer. Pellet the suspension by centrifugation at 100 x g for 7 min, remove the supernatant, and resuspend in complete growth medium.</li>
</ol>
5. Neurosphere Culture
<ol>
	<li>From the cell suspension obtained at isolation, count the number of viable cells using Trypan blue dye exclusion and a hemocytometer.</li>
	<li>Dilute the cell suspension in complete growth medium and seed at 1 x 105 cells/cm2 into uncoated wells of a 6 well plate.</li>
	<li>Incubate at 37 &#176;C, 5% CO2 and &#62; 95% humidity for 14 - 28 days, replace 50% of the growth media every 7 days. Measure the diameter of the neurospheres regularly. If allowed to grow beyond 100 &#181;m in diameter, cell death and spontaneous differentiation can occur within the neurospheres.</li>
</ol>
6. Neurosphere Passage
<ol>
	<li>To passage as a floating culture, when the neurospheres reach approximately 100 &#181;m in diameter, combine all the neurosphere containing media into a single tube. Pellet by centrifugation at 100 x g for 7 min and remove the supernatant.</li>
	<li>Resuspend the pellet in 1 ml of 0.1% Trypsin EDTA and incubate for 7 min in a water bath at 37 &#176;C. Gently pipette up and down 100 times, using a 200 &#181;l pipette tip, to ensure dissociation of the neurospheres.</li>
	<li>Halt the enzyme reaction by adding 5 ml of serum media to the tube and centrifuge at 100 x g for 7 min.</li>
	<li>Remove the supernatant, resuspend the cell pellet in 1 ml complete growth medium and count the number of viable cells using Trypan blue dye exclusion and a hemocytometer.</li>
	<li>Dilute the cell suspension in complete growth medium and seed at 1 x 105 cells/cm2 into uncoated wells of a 6 well plate. Incubate at 37 &#176;C, 5% CO2 and &#62;95% humidity for 14 - 28 days, replacing 50% of the growth media every 7 days.</li>
</ol>
7. Adherent Culture of Neural Precursor Cells
<ol>
	<li>Add a sufficient volume of 0.1% gelatin solution to cover the surface of a tissue culture dish and cure in an incubator at 37 &#176;C for 1 hr prior to processing the neurospheres for adherent culture.</li>
	<li>From the floating neurosphere culture, combine all media into a single tube. Pellet the neurospheres by centrifugation at 100 x g for 7 min and remove the supernatant.</li>
	<li>Resuspend the pellet in 1 ml of 0.1% Trypsin EDTA and incubate for 7 min in a water bath at 37 &#176;C. Gently pipette up and down 100 times, using a 200 &#181;l pipette tip.</li>
	<li>Add 5 ml of serum media to halt the enzyme reaction and centrifuge at 100 x g for 7 min. Discard the supernatant and resuspend the cell pellet in 1 ml complete growth medium and count the number of viable cells using Trypan blue dye exclusion and a hemocytometer.</li>
	<li>Dilute the cell suspension in complete growth medium, remove the gelatin from the tissue culture flasks and seed the cells at 1 x 104 cells/cm2.</li>
	<li>Incubate the cells at 37 &#176;C, 5% CO2 and &#62; 95% humidity. Replace the media with fresh, warmed, complete growth media every three days until the culture reaches approximately 80% confluence (after approximately 7 days).</li>
</ol>
8. Neural Colony Forming Assay
<ol>
	<li>Combine Neural Colony Forming Cell (NCFC) Assay media components: NCFC serum free medium, Proliferation NS-A, and collagen solution (3 mg/ml). Supplement this media with 2 &#181;g/ml heparin, 20 ng/ml EGF, and 10 ng/ml bFGF.</li>
	<li>Following hippocampal tissue dissociation resuspend the cells in neural colony forming assay medium, pre-warmed to 37 &#176;C in a water bath. Seed the cells at a clonal density of 1.1 x 105 cells/ml into a 35 mm culture dish.</li>
	<li>Incubate the cells in this semi-solid collagen matrix at 37 &#176;C, 5% CO2 and &#62; 95% humidity. Culture for 28 days, replacing 50% of the growth media every 7 days.</li>
</ol>
9. Differentiation of Neural Precursor Cells
<ol>
	<li>Add a sufficient volume of 5 &#181;g/cm2 laminin solution to cover the surface of a tissue culture dish and cure in an incubator at 37 &#176;C for 24 hr before processing the cells for differentiation. 
	Note: Remove the laminin solution and rinse the coated surface twice in DPBS immediately prior to seeding.</li>
	<li>When passaging, seed the cell suspension at 1 x 104 cells/cm2 in DMEM-F12 (3:1) media (pre-warmed to 37 &#176;C) supplemented with 20 ng/ml EGF and 40 ng/ml bFGF onto the laminin coated culture dish.</li>
	<li>Incubate the cells at 37 &#176;C, 5% CO2 and &#62; 95% humidity and allow cells to proliferate for 3 days.</li>
	<li>To differentiate the adherent neural precursor cells, after 3 days replace the media with differentiation media containing DMEM-F12 (3:1) media pre-warmed to 37 &#176;C, supplemented with 10 ng/ml brain derived neurotrophic factor (BDNF).</li>
	<li>During differentiation replace 50% of the media every 3 days. Differentiation occurs gradually over approximately 21 - 28 days. 
	Caution: Once the cells have begun to differentiate, only replace 50% of the media at one time as exposure to air can have a detrimental effect on the health of the cells.</li>
</ol>

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M., Conti, L., Sun, Y., Goffredo, D., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+neural+stem+(NS)+cells+from+fetal+and+adult+forebrain.">Adherent neural stem (NS) cells from fetal and adult forebrain.</a> Cereb Cortex. 16, 112-120 (2006).</li><li>Ma, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-extracellular+matrix+interactions+regulate+neural+differentiation+of+human+embryonic+stem+cells.">Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.</a> BMC Dev Biol. 8 (90), 1-13 (2008).</li><li>Goffredo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting+the+conditions+for+efficient,+robust+and+reproducible+generation+of+functionally+active+neurons+from+adult+subventricular+zone-derived+neural+stem+cells.">Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells.</a> Cell Death Differ. 15 (12), 1847-1856 (2008).</li></ol>

The authors have nothing to disclose.

The protocols described here are optimized to maintain favorable culture conditions for maximizing cell viability. The speed and care taken during extraction, isolation and expansion is of critical importance. A critical step for establishing adherent monolayer expansion is the effective dissociation of the primary neurospheres. Following passage, insufficiently dissociated neurospheres may generate secondary floating neurospheres. During media change, these neurospheres may be removed, dissociated and reseeded for adherent monolayer passage. Conversely, if post-dissociation cell viability is below 80%, this may be indicative of excessive pipetting during dissociation. Neural precursor cells, and in particular their differentiated counterparts, are sensitive to changes in pH outside of the physiological range, and exposure to air. The observation of sudden widespread cell death may be attributable to these factors. Consequently, a critical step during media changes is for the media to be made fresh each time, and for only 50% of the volume to be replaced. Finally, while mitogenic factors EGF and bFGF support the survival and proliferation of hippocampal neural precursors under serum-free conditions in vitro22,23, the cellular response to these mitogens is highly dependent on the cell developmental age and mitogenic concentration24,25. Therefore, ensuring minimal experimental variation in these components is crucial for generating consistent, reproducible cell cultures.
For maximum viability, cells should be seeded for culture within 6 hours post mortem. However, the extracted brain may be temporarily stored in PBS at 4 &#176;C. This modification may allow for isolation to be delayed by up to 24 hours with minimal reduction in cell yield26,27. Growth factor supplementation of the culture media may also be modified to enhance proliferation or to encourage the differentiation of specific neural cell types. Insulin-like growth factor 1 (at 100 ng/ml) has been shown to have a supportive effect on rodent neural stem cells in vitro28,29, while under differentiation conditions pro-neuronal factor BDNF30 may be used in conjunction with factors such as Interleukin 7 (at 500 ng/ml) or retinoic acid (at 0.1 &#181;M) to induce a bias towards neuronal subtype or glial differentiation31,32.
The decision on whether to expand neural precursor cells as floating neurospheres or as an adherent monolayer depends largely on the desired downstream applications and culture characteristics. While the neurosphere culture system is simplistic and highly reproducible, it is limited in its ability to generate homogeneous populations of cells. The complex microenvironment within each sphere can promote apoptosis and spontaneous differentiation, encouraging a heterogeneous population33, while reported fusion of neurospheres that can confound quantification of neural precursor cell populations34. Moreover, the repeated mechanical dissociation required for neurosphere passage may also lead to detrimental stress, senescence or even cell death. The use of alternative dissociation enzyme, such as papain, may affect the resultant cell viability16. Conversely, the adherent monolayer culture system, with more uniform exposure to environmental mitogens, encourages greater homogeneity in cell type and more symmetrical division. This system has been shown to produce a niche independent population of cells, based on the expression of key stem cell markers35,36. This system requires the use of pre-coated culture vessels to promote cellular adherence. The choice of culture substrate should be carefully considered, as it may have significant effects on the differentiation profile of the cultured neural precursors25,37.
Here we present effective protocols for the isolation, expansion and differentiation of adult canine hippocampal neural precursor cells from fresh post mortem tissue. Adult canine neural precursors are under-represented in the literature18; with complete protocols for their isolation and expansion, even less so17. Our protocols may then serve to increase awareness of this valuable animal model and represent a significant addition to this modest number of studies. Of significance, using our unique method, adult canine hippocampal neural precursors can be successfully expanded more than 10 population doublings. A similar study using adult canine hippocampal neural precursor reported that larger neurospheres, passaged at 100 - 150 &#181;m in diameter, ceased proliferation beyond the fifth generation17. As noted in our protocol, excessive neurosphere growth can encourage spontaneous differentiation and apoptosis, which over serial passage may have led to this reduced proliferative ability. We also provide a protocol for adherent monolayer expansion of this cell population, as an alternative to the classical neurosphere expansion system17-21. This alternative system affords the user more control over the cellular environment, and significantly broadens the range of downstream applications for these cells, with potential for phenotypic homogenization and directed neuronal differentiation35,38.
Cultured adult canine hippocampal neural precursor cells have applications across a wide range of cellular and neurobiological research fields. The canine brain possesses a closer homology to the human brain than existing rodent models. As such, adult canine neural precursor cells represent an important, yet understudied, resource. These cells may be used to gain further insight into the mechanisms behind adult neurogenesis in humans, and for the development of regenerative therapies that target this process.

Regional variations in the rate of neurogenesis have been observed along the dorsoventral axis of the rodent hippocampus1,2. Furthermore, the rates of hippocampal neurogenesis also show distinct inter-species variation, with precursor cell turnover in the subgranular zone shown to be significantly lower in adult humans than in rodents3-5. Inter-species differences in hippocampal structural anatomy may be relevant here, as it has been postulated that neural stem cell distribution along the murine ventricular neuraxis may be influenced by cephalic flexures during embryological development6. To date, the rodent brain remains the most popular system in which to study adult neurogenesis. However, the brain of the domestic dog (Canis familiaris), with a size and structural organization intermediate between that of humans and rodents7, represents a valuable yet highly underused animal model. The canine hippocampus in particular embodies this structurally intermediate nature8-10 and can provide a unique perspective on intrinsic variations in neural precursor cell populations. With many closer parallels to the human brain, the canine model may also offer insight into the biology of adult human neurogenesis.
In vitro culture assays have become a key tool for the study of neural precursors and the cellular and biomolecular processes of adult neurogenesis. The neurosphere assay and adherent monolayer culture represent the two predominant systems for expanding neural precursor cells in vitro11-13. Protocols for brain extraction, hippocampal microdissection or neural precursor culture assays have been well documented for the rodent model14-16. However, for the adult canine brain they remain comparatively few17,18, focused instead on fetal or neonatal tissue19-21.
In our published study7 we investigated regional variations in neurogenesis and neural precursor cell populations across the dorsoventral axis of the adult canine hippocampus. Although highly dependent on breed, adulthood in canines is reached between 1 and 3 years of age. Here, we present detailed methods for the extraction, isolation and culture of neural precursor cells from the canine hippocampus. We provide systematic protocols for the expansion of neural precursor cells as both floating neurospheres and as an adherent monolayer culture, and for their subsequent differentiation into mature neural cell types.

<table><tbody><tr><td>1,000 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF1000</td><td></td></tr><tr><td>150 mm Petri dish</td><td>BD Biosciences</td><td>351058</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Greiner Bio One</td><td>188271</td><td></td></tr><tr><td>200 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF200</td><td></td></tr><tr><td>24 well culture plate</td><td>Greiner Bio One</td><td>662160</td><td></td></tr><tr><td>35 mm tissue culture dish</td><td>BD Biosciences</td><td>353001</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>6 well culture plate</td><td>BD Biosciences</td><td>351146</td><td></td></tr><tr><td>B-27 Supplement (50x) serum free</td><td>Life Technologies&amp;nbsp;</td><td>17504044</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td>Life Technologies</td><td>13256029</td><td></td></tr><tr><td>Brain derived neurotrophic factor (BDNF)</td><td>Millipore</td><td>GF029</td><td></td></tr><tr><td>Collagen solution</td><td>Stem Cell Technologies</td><td>04902</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>DMEM (4.5 g/L, D-glucose) 500 ml</td><td>Life Technologies</td><td>11960044</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>BD Biosciences</td><td>354001</td><td></td></tr><tr><td>F-12 nutrient mixture (Ham) (1x) Liquid</td><td>Life Technologies</td><td>31765035</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Life Technologies</td><td>16141079</td><td></td></tr><tr><td>Gelatin from Porcine Skin Type A</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>L-alanyl-L-glutamine dipeptide (GlutaMAX)</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Heparin sodium salt from (porcine)</td><td>Sigma-Aldrich</td><td>H314950KU</td><td></td></tr><tr><td>Laminin (mouse)</td><td>Life Technologies</td><td>23017015</td><td></td></tr><tr><td>NCFC serum free medium (NeuroCult)</td><td>Stem Cell Technologies</td><td>5720</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>Proliferation NS-A (NeuroCult)</td><td>Stem Cell Technologies</td><td>05773</td><td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&amp;nbsp; from Stem Cell Technologies.</td></tr><tr><td>NSC basal medium (Rat; NeuroCult)</td><td>Stem Cell Technologies</td><td>5770</td><td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&amp;nbsp;</td></tr><tr><td>Penicillin/Streptomycin (5,000 U/ml)</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>Povidone-iodine</td><td>Munipharma</td><td>Betadine</td><td></td></tr><tr><td>Trypan blue (0.4%)</td><td>Life Technologies</td><td>15250061</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>Class II biological safety cabinet</td><td>ThermoFisher Scientific</td><td>Safe 2020 1.2</td><td></td></tr><tr><td>Brain knife (disposable)</td><td>Macroknife</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td>ThermoFisher Scientific</td><td>HERAcell 150i</td><td></td></tr><tr><td>Centrifuge</td><td>Hettich</td><td>Universal 320R</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Dumont</td><td></td><td></td></tr><tr><td>Easypet Electric pipette</td><td>Eppindorf&amp;nbsp;</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>Boeco</td><td>Bright-Line Improved Neubauer</td><td></td></tr><tr><td>Manual pipettes</td><td>Eppindorf research</td><td></td><td></td></tr><tr><td>Oscillating bone saw&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Scalpel blades (No.21)</td><td>Paramount</td><td></td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Delta</td><td></td><td></td></tr><tr><td>Water bath</td><td>Grant</td><td>JB Aqua 18 plus</td><td></td></tr></tbody></table>

isolation and expansion of adult canine hippocampal neural precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

Through the use of in vitro neural precursor assays, neurogenesis and neural precursor cell populations were characterized and compared across the dorsoventral axis of the adult canine hippocampus. Neural precursor cells derived from isolated hippocampal tissue formed floating neurospheres within 14 days of isolation, reaching a diameter of 100 &#181;m by 28 days of culture. Neurospheres derived from dorsal and ventral isolates showed no difference in mean size, and following enzymatic dissociation into single cells, could be passaged as floating neurosphere cultures. Secondary floating neurospheres from both hippocampal regions were able to form within 5 days from passage. When seeded at 1 x 104 cells/cm2 onto 0.1% gelatin coated culture flasks, neural precursor cells were also able to proliferate as adherent monolayers (Figure 1A). No morphological differences were observed between dorsal and ventral hippocampal neural precursor cells and both adherent cultures were able to undergo over 10 population doublings without any observed slowing in passage-time (Figure 1B). Nestin and Sox2 neural stem cell gene expression in adherent culture was not significantly different across the hippocampal dorsoventral axis (Figure 2). More extensive characterization of gene and protein expression, confirming the neural precursor identity of these cells, is reported in our published data7.
Using an adapted Neural Colony Forming assay4,6 we assessed neural precursor cell frequency in order to quantify potential differences in neural precursor cell populations across the dorsal and ventral subregions of the canine hippocampus. Cells were seeded at clonal density in a semi-solid collagen matrix that precludes cell fusion, and cultured for 28 days (Figure 3A). Within subject comparisons revealed a significantly greater frequency of larger spheres per unit area in dorsal (7.3 &#177; 2.0) compared to ventral hippocampal-derived cultures (3.6 &#177; 1.7; n = 5; repeated measures ANOVA F = 96.8; p = 0.001; Figure 3B).
Under differentiation conditions, adherent canine neural precursor cells showed progressive alterations in gross morphology, developing longer and more elaborate processes. Protein expression for neuronal (&#946;III-tubulin) and glial (Glial fibrillary acidic protein; GFAP) markers also increased following differentiation (Figure 4). No significant difference observed in the number of positively labeled cells between dorsal and ventral isolates. Our previously published data corroborates these protein expression changes, demonstrating up-regulation of their associated genes along with down-regulation of neural precursor genes over the 28 day differentiation period7.
<img alt="Figure 1" src="/files/ftp_upload/54953/54953fig1.jpg" /> 
Figure 1: In Vitro Expansion of Adult Canine Hippocampal Neural Precursor Cells. Adult canine hippocampal neural precursor cells isolated from the dorsal and ventral hippocampal regions are expanded as (A) floating neurospheres or as (B) an adherent monolayer. (C) As an adherent monolayer these neural precursor cells could undergo passage over 10 times without significant decline in doubling speed. n = 5; scale = 50 &#181;m. Modified from published figure7. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54953/54953fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54953/54953fig2.jpg" /> 
Figure 2: Gene Expression in Proliferating Adult Canine Hippocampal Neural Precursor Cells. Expression of neural stem cell genes (A) Nestin and (B) Sox2 was confirmed in adult canine hippocampal neural precursor cells under adherent proliferative culture conditions. Expression of these genes was equivalent in both the dorsal and ventral hippocampus derived cells. Adult canine fibroblasts were used as a control line to compare differences in relative gene expression. n = 5; error bars = SEM. Modified from published figure7. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54953/54953fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54953/54953fig3.jpg" /> 
Figure 3: Neural Colony Forming Assay. (A) Adult canine neural precursor cells, seeded at clonal density into a semi-solid collagen matrix, form neurospheres within 28 days of culture. (B) Neural precursor cells derived from the dorsal hippocampus generate significantly greater numbers of neurospheres per unit area than those derived from the ventral hippocampus. n = 5; error bars = SEM; scale = 50 &#181;m. Modified from published figure7. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54953/54953fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54953/54953fig4.jpg" /> 
Figure 4: Immunocytochemistry of Differentiated Adult Canine Hippocampal Neural Precursor Cells. When cultured in BDNF for 28 days, adult canine neural precursors from both the dorsal and ventral hippocampus differentiate into mature neuronal (&#946;III-tubulin positive) and glial (GFAP positive) cell types. n = 5; scale = 50 &#181;m. Modified from published figure7. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54953/54953fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

isolation-and-expansion-of-adult-canine-hippocampal-neural-precursors

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.0K Views.  University of Sydney.  De hond hersenen is een waardevol model waarin volwassen neurogenese bestuderen. hier gepresenteerde zijn protocollen voor het isoleren en het uitbreiden van volwassen honden hippocampus neurale voorlopercellen uit primaire hersenweefsel. 

Video: Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

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 ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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 ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

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.

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 ...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

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