Name: isolation and expansion of adult canine hippocampal neural precursors
Uploaded: 2016-11-29T15:00:00
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).

I overensstemmelse med New South Wales, Australien lov, post mortem hjernevæv blev erhvervet fra voksne hunde aflivet af årsager relateret til undersøgelsen. 1. Fremstilling af Culture Medium <ol><li> Der fremstilles en 0,1% gelatine opløsning ved tilsætning af 0,1 g gelatine pulver til 100 ml destilleret vand og omrøre ved 37 ° C indtil opløsning. Steriliser opløsningen ved UV-bestråling i 15 minutter. Dette medie kan derefter opbevares ved 4 ° C i op til 1 måned. </li><li> Der fremstille...

<ol>
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S., 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=Neurogenesis+in+the+adult+human+hippocampus.">Neurogenesis in the adult human hippocampus.</a> Nat Med. 4 (11), 1313-1317 (1998).</li><li>Bull, N. D., Bartlett, P. 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A., 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=Isolation+and+transplantation+of+multipotential+populations+of+epidermal+growth+factor-responsive,+neural+progenitor+cells+from+the+canine+brain.">Isolation and transplantation of multipotential populations of epidermal growth factor-responsive, neural progenitor cells from the canine brain.</a> J Neurosci Res. 50 (5), 862-871 (1997).</li><li>Ray, J., Peterson, D. A., Schinstine, M., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+differentiation,+and+long-term+culture+of+primary+hippocampal+neurons.">Proliferation, differentiation, and long-term culture of primary hippocampal neurons.</a> Proc. Natl. Acad. 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E., 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=IGF-1+acts+as+controlling+switch+for+long-term+proliferation+and+maintenance+of+EGF/FGF-responsive+striatal+neural+stem+cells.">IGF-1 acts as controlling switch for long-term proliferation and maintenance of EGF/FGF-responsive striatal neural stem cells.</a> Int. J. Med. Sci. 10 (5), 522-531 (2013).</li><li>Ahmed, S., Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+enhances+the+differentiation+but+not+the+survival+of+CNS+stem+cell-derived+neuronal+precursors.">BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors.</a> J Neurosci. 15 (8), 5765-5778 (1995).</li><li>Wohl, C. 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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>

De her beskrevne protokoller er optimeret til at fastholde gunstige dyrkningsbetingelser for maksimering af cellelevedygtighed. Hastigheden og omhu under ekstraktion, isolering og ekspansion er af afgørende betydning. Et afgørende skridt til etablering af vedhæftende monolag ekspansion er den effektive adskillelse af de primære neurosfærer. Efter passage, utilstrækkeligt dissocierede neurosfærer kan generere sekundære flydende neurosfærer. Under medier forandring, kan disse neurosfærer fjernes, adskilles og ge...

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.

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			<td height="21" style="height:21px;width:337px;">1000 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF1000</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm petri dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL centrifuge tubes</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well culture plate</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm tissue culture dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 &#181;m cell strainer</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well culture plate</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">B-27 Supplement (50X) serum free</td>
			<td style="width:196px;">Life Technologies&#160;</td>
			<td style="width:252px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Basic fibroblast growth factor (bFGF)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">13256029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain derived neurotrophic factor (BDNF)</td>
			<td style="width:196px;">Millipore</td>
			<td style="width:252px;">GF029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (4.5g/L, D-glucose) 500mL</td>
			<td style="width:196px;">Life Technologies&#160;</td>
			<td style="width:252px;">11960044&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">DPBS</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal growth factor (EGF)</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">354001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F-12 nutrient mixture (Ham) (1X) Liquid</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">31765035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">16141079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from Porcine Skin Type A</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-alanyl-L-glutamine dipeptide (GlutaMAX)</td>
			<td>Life Technologies</td>
			<td style="width:252px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt from (porcine)</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">H314950KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin (mouse)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">23017015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proliferation NS-A (NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">05773</td>
			<td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&#160; from Stem Cell Technologies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NSC basal medium (Rat; NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">5770</td>
			<td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Penicillin/Streptomycin (5000 U/mL)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-iodine</td>
			<td style="width:196px;">Munipharma</td>
			<td style="width:252px;">Betadine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Trypan blue (0.4%)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Class II biological safety cabinet</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:252px;">Safe 2020 1.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain knife (disposable)</td>
			<td style="width:196px;">Macroknife</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Cell culture incubator</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:252px;">HERAcell 150i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Centrifuge</td>
			<td style="width:196px;">Hettich</td>
			<td style="width:252px;">Universal 320R</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Forceps</td>
			<td style="width:196px;">Dumont</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Easypet Electric pipette</td>
			<td style="width:196px;">Eppindorf&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:196px;">Boeco</td>
			<td style="width:252px;">Bright-Line Improved Neubauer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Manual pipettes</td>
			<td style="width:196px;">Eppindorf research</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oscillating bone saw&#160;</td>
			<td style="width:196px;"></td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blades (No.21)</td>
			<td style="width:196px;">Paramount</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Scissors&#160;</td>
			<td style="width:196px;">Delta</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:196px;">Grant</td>
			<td style="width:252px;">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.

Gennem brug af in vitro neurale precursorceller assays, neurogenese og neurale precursor cellepopulationer blev karakteriseret og sammenlignet hen over dorsoventral akse af den voksne hunde hippocampus. Neurale præcursorceller afledt isoleret hippocampus væv dannet flydende neurosfærer senest 14 dage efter isolation og nåede en diameter på 100 um med 28 dage af kultur. Neurosfærer afledt af dorsale og ventrale isolater viste ingen forskel i den gennemsnitlige størrelse, o...

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

Den hund hjerne er en værdifuld model til at studere voksen neurogenese. Præsenteret her er protokoller til isolering og udvide voksne hunde hippocampus neurale precursorceller fra primær hjernevæv.

Isolering og Udvidelse af Voksen Canine Hippocampale Neural Forstadier

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

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Ex Utero Elektroporering og organotypisk Slice kultur Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Isolering af neurale stam- / progenitorceller fra Periventricular Region Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Stabil og effektiv genetisk modificering af celler i Voksen Mouse V-SVZ til analyse af neural stamcelle selvstyrende og ikke-selvstændige effekter

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Ex Vivo Kultur Chick cerebellare Skiver og Rumligt Målrettet Elektroporation af granulat celleforstadier

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Isolering og dyrkning af Adult Zebrafisk Brain-afledte Neurosfærer

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

Dyrkning Menneskelig pluripotente og neurale stamceller i en Lukket Cell Culture System for Basic og Prækliniske Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Isolation og udvidelse af mesenchymstamceller / stromacellers afledt af humane Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Stimulering af Notch signalering i Mouse osteoklastpræcursorer

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

Isolation og kultur af primær epikardielle celler, der stammer fra menneskelige voksen og fostrets hjerte prøver

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Afledning og differentiering af Canine æggestokkene mesenkymale stamceller

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...