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).

Gemäß New South Wales, Australien Gesetz, post mortem Hirngewebe wurde von erwachsenen Hunden aus Gründen in keinem Zusammenhang mit der Studie euthanasiert erworben. 1. Herstellung von Kulturmedium <ol><li> Bereiten einer 0,1% igen Gelatinelösung durch Zugabe von 0,1 g Gelatinepulver zu 100 ml destilliertem Wasser und Rühren bei 37 ° C bis gelöst. Sterilisieren der Lösung durch UV-Bestrahlung für 15 min. Diese Medien können dann für bis zu 1 Monat bei 4 ° C gelagert werden. </li><li> Vorbereiten eines 3: 1-Verhältnis von Dulbeccos modifiziertem Eagle-Medium (DMEM) und Nährstoffgemisch F12 durch Zugabe von 167 ml F-12 bis 500 ml DMEM (4,5 g / L D-glucose). Hinzufügen 6,7 ml Penicillin / Streptomycin-Lösung (für eine Endkonzentration von 1%). Dieses Medium kann für bis zu 1 Monat bei 4 ° C im Dunkeln gelagert werden. </li><li> Bereiten 500 ml Serum Medien durch die Kombination 440 ml DMEM (4,5 g / L D-glucose), 5 ml L-Alanyl-L-Glutamin-Dipeptid (200 mM) 5 ml Penicillin / Streptomycin und 50 ml fötales Bovine Serum (FBS). Dieses Medium kann für bis zu 1 Monat bei 4 ° C im Dunkeln gelagert werden. </li><li> Komplettes Wachstumsmedium besteht aus Neural Stem Cell (NSC) Basal Medium und Proliferation Neural Supplement-A (NS-A), 2% FBS, 2 ug / ml Heparin, 20 ng / ml epidermaler Wachstumsfaktor (EGF) und 10 ng / ml basischer Fibroblasten-Wachstumsfaktor (bFGF). Machen Sie diese Medien frisch, unmittelbar vor der Gehirnsezierung, und warm bis 37 ° C in einem Wasserbad. </li></ol> 2. Gehirn Extraction <ol><li> Beginnen Extraktion innerhalb von 6 Stunden post mortem durch die dorsale Oberfläche des Kopfes des Hundes Sterilisieren mit PVP-Jod. </li><li> Mit einem Skalpell machen einen Längsschnitt entlang der sagittalen Mittellinie über die gesamte Rückenfläche des Schädels, die zugrunde liegenden Kaumuskeln zu belichten. Die Länge dieser Schnitt wird von der Art und Alter des Hundes ab. </li><li> An den rostralen und kaudalen Grenzen des Schädels, machen zusätzliche 5 - 10 cm senkrecht Schnitte auf beiden Seiten, übergebening hinter den Augen und Ohren sind, zu ermöglichen, die Haut vollständig reflektiert werden. </li><li> Mit einem Skalpell, trennen Sie die Kaumuskeln aus ihrer Befestigung an der Sagittalkamm des Schädels und kratzen alle verbleibenden Bindegewebe aus dem Schädel. </li><li> ein oszillierendes Knochen Mit sah die Kappe des Schädels in einer Umfangslinie geschnitten. Mit Hilfe einer Sonde oder einem Skalpell durchtrennen alle verbleibenden Anlagen mit der Dura, und dann vorsichtig die Schädelkappe entfernen Sie die dorsale Oberfläche des Gehirns aufzudecken. Achtung: Die Dicke des Knochens variiert in den verschiedenen Regionen in den Schädel, so dringen Vorsicht geboten ausgeübt werden , um nicht zu tief und das darunter liegende Gehirn schädigen. </li><li> Sever das Rückenmark durch ein Skalpell zwischen der oberen Halswirbel stecken. </li><li> Mit der einen Hand, um das Gehirn sanft heben, verwenden Sie ein Skalpell vorsichtig auf das Gehirn aus dem Schädelgrube befreien und durchtrennen die Verbindungs ​​Nerven und Blutgefäße befindet sich an seiner Bauchseite. Heben Sie die bregen aus dem Schädel und in einen Behälter mit DPBS. Achtung: Die großen Riechkolben des Hunde-Gehirn tief in die vordere Schädelgrube erstrecken kann. Es muss darauf geachtet werden, wenn das Gehirn zu entfernen, um diese Strukturen zu erhalten. </li></ol> 3. Hippocampale Dissection <ol><li> Spülen Sie das Gehirn in Dulbeccos phosphatgepufferter Salzlösung (DPBS) kein Blut zu entfernen und sie Rückenseite nach unten auf eine 150 mm Petrischale. </li><li> bisect langsam das Gehirn längs durch die Mitte Sagittalebene einen nassen Gehirn Messer und eine einzelne Scheibe verwenden, die über die gesamte Länge der Klinge verwendet. Wenden Sie keine zusätzlichen Abwärtsdruck, oder eine Sägebewegung, da dies das Gewebe beschädigen können. </li><li> Platzieren jeder Hemisphäre medial Fläche nach oben, sezieren aus dem Hippocampus eines Skalpells und einer feinen Pinzette verwenden und übertragen sie auf eine 35-mm-Gewebekulturschale. </li></ol> 4. Isolierung von neuralen Vorläuferzellen <ol><li> Mince die tAusgabe Proben mit einem Skalpell in ca. 1 mm 3 Stück verwenden. </li><li> Übertragen Sie das Gewebe in einen 15-ml-Röhrchen, 2 ml 0,1% Trypsin-EDTA, und Inkubation für 7 Minuten in einem Wasserbad bei 37 ° C. </li><li> Halt die Enzymreaktion durch Zugabe von 4 ml von Serum Medien auf das Rohr. Pellet die Suspension durch Zentrifugation bei 100 g für 7 min und entfernen Sie den Überstand. </li><li> Resuspendierte das Pellet in 300 ul DPBS und mechanisch durch vorsichtiges Pipettieren bis distanzierte und unten, eine 1000 ul Pipettenspitze, bis eine glatte Homogenat Formen. </li><li> In 14 ml Serum Medien in das Röhrchen und übergeben Sie die Zellsuspension durch ein Sieb 40 &amp; mgr; m-Zelle. Pellet die Suspension durch Zentrifugation bei 100 xg für 7 min, um den Überstand zu entfernen, und resuspendieren in komplettem Wachstumsmedium. </li></ol> 5. Neurosphäre Kultur <ol><li> Von der Zellsuspension bei Isolierung erhalten, zählen die Anzahl der lebenden Zellen Trypanblaufarbstoff Ausschluss verwenden und eine HämoZytometer. </li><li> Verdünne die Zellsuspension in komplettem Wachstumsmedium und Samen bei 1 x 10 5 Zellen / cm2 in unbeschichteten Vertiefungen einer Platte mit 6 Vertiefungen. </li><li> Inkubieren bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit für 14 - alle 7 Tage 28 Tage 50% der Wachstumsmedien ersetzt. Messen Sie den Durchmesser der regelmäßig Neurospheres. Wenn erlaubt über 100 &amp; mgr; m im Durchmesser zu wachsen, Zelltod und spontane Differenzierung in den Neurosphären auftreten. </li></ol> 6. Neurosphäre Passage <ol><li> Um Passage als schwimmender Kultur, wenn die Neurosphären etwa 100 &amp; mgr; m im Durchmesser erreichen, kombinieren alle Neurosphere haltigen Medien in einem einzigen Rohr. Pellet durch Zentrifugation bei 100 g für 7 min und entfernen Sie den Überstand. </li><li> Resuspendieren des Pellets in 1 ml 0,1% Trypsin EDTA und Inkubation bei 37 ° C für 7 min in einem Wasserbad. Pipette vorsichtig nach oben und unten 100-mal, eine 200 ul Pipettenspitze, um sicherzustellen, dissociatiauf der Neurosphären. </li><li> Halt die Enzymreaktion durch Zugabe von 5 ml Serum Medien in das Röhrchen und zentrifugiert bei 100 g für 7 min. </li><li> Entfernen Sie den Überstand, Zellpellet in 1 ml komplettem Wachstumsmedium und zählen die Anzahl der lebenden Zellen Trypanblaufarbstoff Ausschluss Verwendung und einer Zählkammer. </li><li> Verdünne die Zellsuspension in komplettem Wachstumsmedium und Samen bei 1 x 10 5 Zellen / cm2 in unbeschichteten Vertiefungen einer Platte mit 6 Vertiefungen. Inkubieren bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit für 14 bis 28 Tage 50% der Wachstumsmedien alle 7 Tage ersetzt werden . </li></ol> 7. adhärenten Kultur von neuralen Vorläuferzellen <ol><li> Um ein ausreichendes Volumen von 0,1% igen Gelatinelösung, die Oberfläche einer Gewebekulturschale und Heilung in einem Inkubator bei 37 ° C für 1 h vor der Verarbeitung des Neurosphären für adhärente Kultur zu decken. </li><li> Von der schwimmenden Neurosphären Kultur, verbinden alle Medien in einem einzigen Rohr. Pellet die neurospheres durch Zentrifugation bei 100 xg für 7 min und den Überstand entfernen. </li><li> Resuspendieren des Pellets in 1 ml 0,1% Trypsin EDTA und Inkubation bei 37 ° C für 7 min in einem Wasserbad. Pipette vorsichtig auf und ab 100 Mal, mit einer 200 ul Pipettenspitze. </li><li> 5 ml Serum-Medien für 7 min, um die Enzymreaktion und Zentrifuge bei 100 xg zu stoppen. Überstand verwerfen und Zellpellet in 1 ml komplettem Wachstumsmedium und zählen die Anzahl der lebenden Zellen Trypanblaufarbstoff Ausschluss Verwendung und einer Zählkammer. </li><li> Verdünnen Sie die Zellsuspension in komplettem Wachstumsmedium, entfernen Sie die Gelatine aus den Gewebekulturflaschen und Samen der Zellen bei 1 x 10 4 Zellen / cm 2. </li><li> Inkubieren der Zellen bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit. Ersetzen Sie die Medien mit frischen, erwärmt, vollständige Wachstumsmedien alle drei Tage, bis die Kultur etwa 80% Konfluenz erreicht (nach ca. 7 Tage). </li></ol> 8. Neural Colony Forming Assay <ol><li> Kombinieren Neural Colony Forming Zelle (NCFC) Assay Medienkomponenten: NCFC serumfreien Medium, Proliferation NS-A und Kollagenlösung (3 mg / ml). Nachtrag: Dieses Medium mit 2 ug / ml Heparin, 20 ng / ml EGF und 10 ng / ml bFGF. </li><li> Hippokampus-Gewebedissoziation Nach Resuspendieren der Zellen in neurale koloniebildenden Assay-Medium, vorgewärmt auf 37 ° C in einem Wasserbad. Samen der Zellen bei einer klonalen Dichte von 1,1 x 10 5 Zellen / ml in eine 35 mm Kulturschale. </li><li> Inkubieren der Zellen in diesem halbfesten Kollagen - Matrix bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit. Kultur für 28 Tage, 50% des Wachstumsmediums alle 7 Tage ersetzt werden. </li></ol> 9. Differenzierung von neuralen Vorläuferzellen <ol><li> Um ein ausreichendes Volumen von 5 &amp; mgr; g / cm 2 Laminin Lösung , um die Oberfläche einer Gewebekulturschale und Heilung in einem Inkubator bei 37 ° C für 24 h zu decken , bevor die Zellen für die Verarbeitung differentiation. Hinweis: Entfernen Sie die Laminin - Lösung und spülen Sie die beschichtete Oberfläche zweimal in DPBS unmittelbar vor der Aussaat. </li><li> Wenn Passagierung Saatgut der Zellsuspension mit 1 x 10 4 Zellen / cm 2 in DMEM-F12 (3: 1) Medium (vorgewärmt auf 37 ° C) mit 20 ng / ml EGF und 40 ng / ml bFGF auf die Laminin beschichteten Kulturschale. </li><li> Inkubieren der Zellen bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit und erlauben Zellen für 3 Tage zu vermehren. </li><li> Um die anhaftende neurale Vorläuferzellen differenzieren, nach 3 Tagen das Medium mit Differenzierungsmedium, das DMEM-F12 ersetzen (3: 1) Medium vorgewärmt auf 37 ° C, supplementiert mit 10 ng / ml brain derived neurotrophic factor (BDNF). </li><li> alle 3 Tage während der Differenzierung 50% der Medien zu ersetzen. Die Differenzierung erfolgt schrittweise über etwa 21 bis 28 Tagen haben. Achtung: Sobald die Zellen zu differenzieren begonnen, ersetzen nur 50% der Medien an einem time als Zutritt von Luft kann eine nachteilige Wirkung auf die Gesundheit der Zellen haben. </li></ol>

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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=Retinoic+acid+enhances+neuronal+proliferation+and+astroglial+differentiation+in+cultures+of+CNS+stem+cell-derived+precursors.">Retinoic acid enhances neuronal proliferation and astroglial differentiation in cultures of CNS stem cell-derived precursors.</a> J Neurobiol. 37 (2), 281-290 (1998).</li><li>Araujo, D. M., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+effects+of+interleukin-4,-7+and-8+on+hippocampal+neuronal+cultures:+potential+involvement+of+glial-derived+factors.">Trophic effects of interleukin-4,-7 and-8 on hippocampal neuronal cultures: potential involvement of glial-derived factors.</a> Brain Res. 600 (1), 49-55 (1993).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Singec, I., 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=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nat Methods. 3 (10), 801-806 (2006).</li><li>Conti, L., 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=Niche-independent+symmetrical+self-renewal+of+a+mammalian+tissue+stem+cell.">Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.</a> PLoS Biol. 3 (9), 283 (2005).</li><li>Pollard, S. 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.

Die Protokolle werden hier beschrieben, optimiert für die Maximierung der Zelllebensfähigkeit günstige Kulturbedingungen zu halten. Die Geschwindigkeit und die Sorgfalt bei der Extraktion, Isolierung und Expansion von entscheidender Bedeutung. Ein entscheidender Schritt für adhärenten einschichtigen Expansion Gründung ist die effektive Spaltung der primären Neurosphären. Nach Durchgang distanzierte unzureichend Sekundär schwebenden Neurosphären Neurosphären erzeugen kann. Während Medienwechsel können diese ...

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.

Durch die Verwendung von in vitro - Assays neuralen Vorläufer, Neurogenese und Populationen neuralen Vorläuferzelle wurden über die dorsoventral Achse des erwachsenen Hippokampus canine charakterisiert und verglichen. Neurale Vorläuferzellen aus isolierten Hippocampus-Gewebe abgeleitet gebildet Floating Neurosphären innerhalb von 14 Tagen der Isolation, die einen Durchmesser von 100 &amp; mgr; m von 28 Tagen der Kultur zu erreichen. abgeleiteten Neurosphären aus dorsalen u...

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

Der Eckzahn Gehirn ist ein wertvolles Modell, bei dem die adulte Neurogenese zu studieren. Präsentiert hier sind Protokolle zur Isolierung und erweitert erwachsenen Hunde-Hippocampus-neuralen Vorläuferzellen aus primären Hirngewebe.

Isolierung und Expansion von adulten Canine Hippocampus Neural Precursors

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

Research

JoVE Journal

Developmental Biology

7.0K Aufrufe.  University of Sydney.  Der Eckzahn Gehirn ist ein wertvolles Modell, bei dem die adulte Neurogenese zu studieren. Präsentiert hier sind Protokolle zur Isolierung und erweitert erwachsenen Hunde-Hippocampus-neuralen Vorläuferzellen aus primären Hirngewebe. 

Serie: 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.

Wachstum und Differenzierung von adulten Hippocampus 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 ...

Forschung

Neurowissenschaften

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 und Expansion der erwachsenen Maus Neural Stem Cells mit dem Neurosphäre 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 ...

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.

Bewertung der dendritischen Verzweigung im Gyrus dentatus des Hippocampus-Region an der Maus

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.

Immunhistochemie und Multiple Markierung mit Antikörper aus dem gleichen Host Arten, die Adult Neurogenese Studieren

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.

Zellsortierung von Neural Stamm- und Vorläuferzellen aus dem erwachsenen Maus Subventrikularzone und Live-Bildgebung ihrer Zellzyklus Dynamics

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.

Isolierung und Kultur von adulten neuralen Stammzellen aus der Maus subcallosa Zone

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.

Assaying Circuit Spezifische Regulation von adulthippocampal neuralen Vorläuferzellen

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.

Isolierung und Expansion von Neurosphären aus postnatalen (P1-3) Maus neurogenen Nischen

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

<meta charset="utf8"></head><body>Effiziente Nukleofektion von NSCs, die aus der subventrikulären Zone der Maus isoliert wurden

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.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Verbesserte Nukleofektion für einen hocheffizienten Gentransfer in murinen subventrikulären Zonen abgeleiteten neuralen Stammzellkulturen

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.

Isolierung, Expansion und Nukleofektion von neuralen Stammzellen aus der adulten subventrikulären Zone der Maus

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 ...