Diese Arbeit wird durch die Natural Sciences and Engineering Research Council of Canada (Grant # 249669 und # 482986) und Heart and Stroke Foundation of Canada (Grant # 485508) gefördert. Die Autoren danken Youssef El-Hayek und Dr. Qi Wan für ihre Unterstützung bei der Entwicklung der experimentellen Protokolle.

Alle Verfahren, die Behandlung der Tiere wurden von der University of Toronto Animal Care Committee in Übereinstimmung mit den Richtlinien des Instituts (Protokoll Nr. 20.009.387) zugelassen. Die folgenden Methoden sollten unter Verwendung steriler Instrumente und Techniken in einer Sterilbank, soweit zutreffend. Im Protokoll-Text bezieht sich der Ausdruck &quot;EFH-SFM&quot; zu serumfreiem Medium mit epidermalen Wachstumsfaktor, basischem Fibroblasten-Wachstumsfaktor und Heparin ergänzt. EFH-SFM wird verwendet, wenn die Untersuchung der Galvanotaxis undifferenzierter NPCs, weil diese Mitogene NPCs in ihrem undifferenzierten Zustand 4 zu halten. Bei der Untersuchung der Galvanotaxis NPC-induzierte zu reifen Zelltypen differenzieren, &quot;FBS-SFM&quot; bezieht sich auf serumfreiem Medium mit 1% fötalem Rinderserum ergänzt. FBS fördert die Differenzierung von NSCs in reife neuronale Phänotypen 5. Ein. Isolation und Kultur von neuralen Vorläuferzellen (Nichtdargestellt im Video) <ol><li> Betäuben ein CD1-Maus (6-8 Wochen alt) mit Isofluran und Opfer über Genickbruch. </li><li> Begießen den Kopf in 70% Ethanol und enthaupten das Tier mit scharfen Dissektion Schere. </li><li> Halten Sie den Kopf mit chirurgischen Pinzette die Haut auf der dorsalen Oberfläche, um den Schädel freizulegen. </li><li> Mit einem Skalpell und nein. 11 Blättern, erzielt das Schädels an der Stirnhöhle entlang der mediolateralen Achse und auch entlang der Sagittalnaht im rostrokaudalen Richtung. </li><li> Schälen Sie die Scheitelbeinen vom Kopf ohne. 7 gebogene Pinzette, dabei nicht zu durchbohren das Hirngewebe. </li><li> Führen Sie einen dünnen Spatel unterhalb des Gehirns ab unter dem Kleinhirn und Weiterentwicklung in Richtung der Riechkolben. Halten Sie den Schädel an Ort und Stelle mit einer Pinzette, ziehen das Gehirn aus dem Schädel und sofort legen Sie sie in eiskaltem künstlichen Liquor (siehe Rezepte unten). </li><li> Unter einem Dissektionsmikroskop, mit sterilenDissektion Schere und Pinzette geschnitten das Gehirn in der Hälfte entlang der Mittellinie. Drehen Sie jeden Hemisphäre, so dass die mediale (cut) Oberfläche nach oben zeigt. </li><li> Wählen Sie eine Halbkugel, und mit der medialen Oberfläche nach oben, suchen Sie die Splenium des Corpus callosum (hinteren Bereich des Corpus callosum). </li><li> Einen Einschnitt von der Oberfläche der Hirnrinde der Splenium des Balkens entlang der dorsoventralen Achse. </li><li> Peel die eingeschnittene Kortex zu der Riechkolben, die medialen und lateralen Wände des lateralen Ventrikel freizulegen. </li><li> Rotieren die Halbkugel so dass der dorsale Oberfläche nach oben zeigt, und mit gekrümmten Mikroschere zum Ausschneiden und sammeln die freiliegenden medialen und lateralen Wänden, die den Bereich, in dem periventrikulären NPCs aufzuhalten 6 enthalten. </li><li> Wiederholen Sie die Schritte 1,8-1,11 für die andere Hemisphäre. </li><li> Pipettieren der isolierten Gewebe in 7 ml Trypsin-Lösung (siehe Rezept unten) in einem 15 ccm Rohr, und legen Sie das Rohr auf einer Wippe in einem 37 ° CInkubator für 25 min. </li><li> Zentrifugieren bei 1.500 rpm für 5 min, den Überstand aspirieren und resuspendieren das Gewebe in 2 ml Trypsin-Inhibitor-Lösung (siehe Rezept unten). </li><li> Gently verreiben das Gewebe mit einer kleinen Bohrung Pasteurpipette 30-50 mal vorsichtig, um Luftblasen zu vermeiden. </li><li> Zentrifugieren bei 1.500 rpm für 5 min, den Überstand aspirieren und resuspendieren in 1-2 ml SFM (Rezept siehe unten) durch Verreiben das Pellet 3-5 mal. </li><li> Zentrifugieren bei 1.500 rpm für 3 min, saugen Sie den Überstand und resuspendieren in 1 ml SFM + EFH. </li><li> Zählen leben Zelldichte mit einem Hämozytometer und Platte die Zellen in einer T25-Kulturkolben in einer Dichte von 10 Zellen pro ul in SFM + EFH. </li><li> Lassen Sie die Kultur für 7 Tage ungestört frei schwebenden primäre Neurosphären von NPCs zusammen ergeben wachsen. </li></ol> 2. Galvanotaxis Chamber Vorbereitung <ol><li> Platz 3 quadratischen Glas-Nr. 1 Deckgläschen (22 x 22 x 0,17 mm) ina Flasche 6N Salzsäure über Nacht. </li><li> Am nächsten Tag, mit einem Diamant-Spitze Glasschneider bis 6 rechteckigen Streifen (22 x 5 x 0,17 mm) aus Glas vom Quadrat nicht geschnitten. 1 Deckgläser. </li><li> Übertragen Sie die acid-washed Platz rutscht und rechteckige rutscht in eine laminare Strömung Kapuze. Waschen Sie die rechteckige und quadratische Streifen zunächst mit 70% Ethanol, dann mit Gewebekultur-grade autoklavierten Wasser, und lassen Sie sie auf einem Kim trocknen Wischen (für zusätzliche Sterilität, kann das Glas an der Luft trocknen erlaubt sein). </li><li> Anwenden Vakuumfett an dem Umfang der einen Fläche der quadratischen Glasobjektträgern und verschließt diese an der Basis von 60 mm Petrischalen aus Kunststoff. </li><li> Anwenden Vakuumfett entlang der langen Achse der einen Oberfläche der rechteckigen Glasstreifen, und schließt diesen an gegenüberliegenden Kanten der quadratischen Glasobjektträgern (derart, dass sie parallel zueinander sind), um eine zentrale Mulde zu schaffen. </li><li> UV-Sterilisation der Kammern für mindestens 15 min in der Sterilbank. </li><li> Pipette 250-300 ul von Poly-L-Lysine auf den zentralen Mulde der Kammern hinzugefügt und bei Raumtemperatur für 2 Stunden. </li><li> Etwa 15 Minuten vor dem Ende der Inkubationszeit, bereiten die Matrigel-Lösung (siehe Rezept unten). </li><li> Saugen Sie die Poly-L-Lysin, waschen Sie die zentralen Tröge mit 1 ml autoklavierten Wasser, und Pipette 250-300 &amp;mgr; l Matrigel Lösung auf den zentralen Tälern. </li><li> Inkubiere die Kammern bei 37 ° C für 1 Stunde. </li><li> Saugen Sie die Matrigel-Lösung und vorsichtig waschen die zentralen Tröge mit 1-2 ml SFM. </li><li> Je 100 ul der EFH-SFM oder FBS-SFM auf den zentralen Tälern und übertragen Sie die Galvanotaxis Kammern auf der Bühne eines Zählen Mikroskop. </li><li> Pipette 3-4 ml des Neurosphäre enthaltenden Kultur in eine 60 mm Petrischale und übertragen die Petrischale auf der Bühne des Mikroskops gezählt. </li><li> Bei einer Betrachtung Ziel 5x, verwenden Sie eine P10 Pipette 5-8 gesamten Neurosphären (bis zu vier gleichzeitig) auf den zentralen Mulde jedes Galvanotaxis übertragenKammer ohne distanziert sie, und sorgfältig verteilt die Neurosphären rund um den zentralen Mulde ohne Unterbrechung des Matrigel Substrat. </li><li> Fügen Sie eine weitere 150-200 ul EFH-SFM oder FBS-SFM auf den zentralen Tälern. </li><li> Übertragen der Galvanotaxis Kammern in einem 37 ° C, 5% CO 2, 100% befeuchteten Inkubator für 17 bis 20 h (wenn Analysieren undifferenzierten NPC), damit die Neurospheres, um zu der Matrigel Substrat haften und dissoziieren in einzelne Zellen, wie in Abbildung 1 dargestellt . Wenn Analysieren differenzierten NSCs sollte die Inkubationszeit bis 69-72 h verlängert, um die Differenzierung der Zellen zu ermöglichen. </li></ol> 3. Live Cell Time-Lapse Imaging <ol><li> Lassen Sie das Live Cell Imaging System bei 37 ° C äquilibrieren, 5% CO 2 für mindestens 30 min vor Beginn der Zeitraffer-Aufnahme. </li><li> Schneiden Sie zwei 12 cm lange Stücke von 1 mm Durchmesser Silber Draht, Spule sie von einem Ende, und legen Sie sie in Clorox bLeach für 20 min zu bilden Ag / AgCl Elektroden. </li><li> Übertragen Sie die Galvanotaxis Kammern auf der Bühne eines Zählen Mikroskop und auswählen, welche Kammer wird für Live-Cell-Imaging Migration Analyse auf Grundlage der folgenden Kriterien verwendet werden: i) die Neurosphären sollte fast vollständig dissoziiert in einzelne Zellen und ii) die Zellen besitzen sollte Runde Morphologien mit wenig bis keine Prozesse sich von den Zellkörper. </li><li> Übertragen des ausgewählten Galvanotaxis Kammer in einer Sterilbank, zusammen mit einem getrennten quadratischen Nr. 1 Deckglas und Vakuum Fett. </li><li> Waschen der Deckel erste rutschen mit 70% igem Ethanol, dann mit autoklaviertem Wasser und Anwendung eines Streifens aus Vakuumfett an zwei parallelen Kanten des Deckglases. </li><li> Absaugen Kulturmedien von der zentralen Rinne der Kammer, dann schnell platzieren das Deckglas (Fett-Seite nach unten) auf die Kammer derart, dass das Fett Streifen auf den beiden parallelen rechteckigen Glasstreifen, effektiv eine Dachan die Kammer. </li><li> Je 100 ml frisches EFH-SFM oder FBS-SFM in den zentralen Mulde durch Kapillarwirkung. </li><li> Verwenden Vakuumfett um Grenzen für Pools von Kulturmedien auf jedem Ende der zentralen Rinne zu schaffen, wie in 2 gezeigt. </li><li> Schneiden Sie zwei 15 cm Stücke von PVC-Schlauch, und verwenden Sie einen 10-ml-Spritze mit einer 18-Gauge-Nadel sorgfältig injizieren Agarose-Lösung in den Schlauch, wodurch keine Blasen bilden in den Rohren, und lassen Sie das Gel für 5 min zu festigen. </li><li> Übertragen der Galvanotaxis Kammer zu der lebenden Zellen System, zusammen mit dem Agarosegel Rohren, Ag / AgCl-Elektroden, und ein Paar von leeren 60 mm Petrischalen, die als Reservoire Kulturmedien verwendet werden und wird die Ag / AgCl-Elektroden enthalten. Lassen Sie die Galvanotaxis Kammer innerhalb des 37 ruhen ° C, 5% CO 2-Umgebung für 20-30 min. </li><li> Während dieser Zeit bereiten die Deckel der 2 leere Petrischalen und den Deckel der Petrischale des Galvanotaxis Kammer die durch Bohren von Löchernin sie mit einem Dremel oder ein ähnliches Werkzeug, wie in Abbildung 3 gezeigt. </li><li> Pipette 1-1,5 ml EFH-SFM oder FBS-SFM auf beiden Seiten der zentralen Rinne und 7-8 ml SFM in jedes leere Petrischale. Legen Sie eine Petrischale auf jeder Seite des Galvanotaxis Kammer der zentralen Mulde und Ort ein Ag / AgCl-Elektrode in jede Schale. Die Lücke zwischen der Kammer und den Galvanotaxis Petrischalen auf elektrische Kontinuität mit den Agarosegel Brücken zu schaffen, wie in Abbildung 4 dargestellt. </li><li> Schließen Sie die Ag / AgCl-Elektroden mit einem externen Netzteil, mit einem Amperemeter in Reihe, um elektrischen Strom zu messen, und schalten Sie die Stromversorgung. Mit einem Voltmeter, um die Stärke des elektrischen Feldes direkt auf der zentralen Mulde zu messen, und den Ausgangspegel der Stromversorgung bis die gewünschte elektrische Feldstärke erreicht ist (die Assays in diesem Labor durchgeführt nutzen eine dcEF Stärke von 250 mV / mm mit elektrischer Strom zwischen 1 und 1,5 mA). </li><li> Initiate der Zeitraffer-Modul auf dem Live Cell Imaging System und ermöglichen das Experiment, um die gewünschte Menge an Zeit laufen. Nach Abschluss des Tests, fixieren die Zellen in 4% Paraformaldehyd für Standard Immunfärbung Analyse. </li></ol>

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Keine Interessenskonflikte erklärt.

Dieses Protokoll wurde von den etablierten Methoden der früheren Studien 7-9 angepasst. Galvanotactic Kammern kann unter Verwendung einer Vielzahl von verschiedenen Techniken, einschließlich der Errichtung eines separaten Glas gut für Einschluss Zellaussaat oder mit Hilfe von CO 2-Laser-Ablation zur Mikrofabrikation des zentralen Rinne 10,11 werden. Einige Techniken können mehr mühsam oder kostspieliger als andere. Wir haben eine einfache und kostengünstige Konstruktion eines Assay...

<table border="1"><tbody><tr><td height="44"> Bezeichnung des Gegenstands </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td></td><td></td><td></td><td> Neuronale Vorläuferzellen Isolation </td></tr><tr><td> 2 M NaCl </td><td> Sigma </td><td> S5886 </td><td> 11,688 g in 100 ml dH 2 O gelöst </td></tr><tr><td> 1M KCl </td><td> Sigma </td><td> P5405 </td><td> 7,456 g in 100 ml dH 2 O gelöst </td></tr><tr><td> 1M MgCl 2 </td><td> Sigma </td><td> M2393 </td><td> 20,33 g in 100 ml dH 2 O gelöst </td></tr><tr><td> 155 mM NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td> 1,302 g in 100 ml dH 2 O gelöst </td></tr><tr><td> 0,5 M Glucose </td><td> Sigma </td><td> G6152 </td><td> 9,01 g gelöst in 100ml dH 2 O </td></tr><tr><td> 108 mM CaCl 2 </td><td> Sigma </td><td> C7902 </td><td> 1,59 g in 100 ml dH 2 O gelöst </td></tr><tr><td> Penicillin-Streptomycin </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Rinderpankreas Trypsin </td><td> Sigma </td><td> T1005 </td><td></td></tr><tr><td> Sheep Hoden Hyaluronidase </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Kynurensäure </td><td> Sigma </td><td> K3375 </td><td></td></tr><tr><td> Ovomucoid Trypsininhibitor </td><td> Worthington </td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100046 </td><td></td></tr><tr><td> F12 </td><td> Invitrogen </td><td> 21700075 </td><td></td></tr><tr><td> 30% Glucose </td><td> Sigma </td><td> G6152 </td><td></td></tr><tr><td> 7,5% NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td></td></tr><tr><td> 1M HEPES </td><td> Sigma </td><td> H3375 </td><td> 23,83 g in 100 ml dH 2 O gelöst </td></tr><tr><td> L-Glutamin </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> EGF </td><td> Invitrogen </td><td> PMG8041 </td><td> Rekonstituieren in 1 ml Hormon-Mix und Aliquote in 20 ul Einheiten. </td></tr><tr><td> FGF </td><td> Invitrogen </td><td> PHG0226 </td><td> Rekonstituieren in 0,5 ml Hormon-Mix und Aliquote in 20 ul Einheiten. </td></tr><tr><td> Heparin </td><td> Sigma </td><td> H3149 </td><td></td></tr><tr><td> Apo-Transferrin </td><td> R &amp; D Systems </td><td> 3188-AT </td><td> 0,1 g in 4 ml dH 2 0 gelöst </td></tr><tr><td> Putrescin </td><td> Sigma </td><td> P7505 </td><td> Auflösen 9,61 mg in Apo-Transferrin solution </td></tr><tr><td> Insulin </td><td> Sigma </td><td> I5500 </td><td> 25 mg in 0,5 ml 0,1 N HCl und fügen Sie 3,5 ml dH 2 0 </td></tr><tr><td> Selen </td><td> Sigma </td><td> S9133 </td><td></td></tr><tr><td> Progesteron </td><td> Sigma </td><td> P6149 </td><td></td></tr><tr><td> Standard Dissection Werkzeuge </td><td> Fine Science Tools </td><td></td><td></td></tr><tr><td> Dissektionsmikroskop </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> Galvanotaxis Chamber Vorbereitung </td></tr><tr><td> Platz Glasabdeckung Dias </td><td> VWR </td><td> 16004 </td><td></td></tr><tr><td> 6N Salzsäure </td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td> Hochvakuumfett </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> 60 mm Petrischalen </td><td> Fisher Scientific </td><td> 0875713A </td><td></td></tr><tr><td> Poly-L-Lysin </td><td> Sigma </td><td> P4707 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td> Tauwetter und Aliquot in 150 ul-Einheiten </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10082139 </td><td> Nur verwenden, wenn induzieren NPC Differenzierung, ansonsten SFM + EFH Kulturmedien wie oben angegeben </td></tr><tr><td> Zählen Mikroskops </td><td> Olymp </td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> Live Cell Time-Lapse Imaging </td></tr><tr><td> Silberdraht </td><td> Alfa Aesar </td><td> 11434 </td><td></td></tr><tr><td> UltraPure Agarose </td><td> Invitrogen </td><td> 15510-027 </td><td></td></tr><tr><td> Hitze Inactivated FBS </td><td> Sigma </td><td> 16140071 </td><td></td></tr><tr><td> PVC-Schlauch </td><td> Fisher Scientific </td><td> 80000006 </td><td> 3/32 &quot;ID x 5/32&quot; OD </td></tr><tr><td> Bleichmittel </td><td> Clorox </td><td></td><td></td></tr><tr><td> 10-ml-Spritze </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 18-Gauge-Nadel </td><td> BD </td><td> 305195 </td><td></td></tr><tr><td> Dremel Bohrmaschine </td><td> Dremel </td><td> Modell 750 </td><td></td></tr><tr><td> Inverses Mikroskop mit befeuchteten, bebrütete Kammer ausgestattet </td><td> Zeiss </td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td> Rezepte <table border="1"><tbody><tr><td> Artikel </td><td> Volumen </td></tr><tr><td> 2 M NaCl </td><td> 6,2 ml </td></tr><tr><td>1M KCl </td><td> 0,5 ml </td></tr><tr><td> 1M MgCl 2 </td><td> 0,32 ml </td></tr><tr><td> 155mm NaHCO 3 </td><td> 16,9 ml </td></tr><tr><td> 1M Glucose </td><td> 1 ml </td></tr><tr><td> 108 mM CaCl 2 </td><td> 0,09256 ml </td></tr><tr><td> Penicillin-Streptomycin </td><td> 1 ml </td></tr><tr><td> Wasser autoklaviert </td><td> 74 ml </td></tr></tbody></table> Künstliche Zerebrospinalflüssigkeit <table border="1"><tbody><tr><td> Artikel </td><td> Volumen oder Masse </td></tr><tr><td> Künstliche Zerebrospinalflüssigkeit </td><td> 30 ml </td></tr><tr><td> Rinderpankreas Trypsin </td><td> 40 mg </td></tr><tr><td> Sheep Hoden Hyaluronidase </td><td> 22,8 mg </td></tr><tr><td> Kynurensäure </td><td> 5 mg </td></tr></tbody></table> &lt;strong&gt; Trypsin-Lösung <table border="1"><tbody><tr><td> Artikel </td><td> Volumen oder Masse </td></tr><tr><td> SFM </td><td> 15 ml </td></tr><tr><td> Ovomucoid Trypsininhibitor </td><td> 10 mg </td></tr></tbody></table> Trypsininhibitorlösung <table border="1"><tbody><tr><td> Artikel </td><td> Volumen </td></tr><tr><td> Wasser autoklaviert </td><td> 37 ml </td></tr><tr><td> 10X DMEM/F12 </td><td> 10 ml </td></tr><tr><td> 30% Glucose </td><td> 2 ml </td></tr><tr><td> 7,5% NaHCO 3 </td><td> 1,5 ml </td></tr><tr><td> 1M HEPES </td><td> 0,5 ml </td></tr><tr><td> Transferrin, Putrescin-Lösung </td><td> 4 ml </td></tr><tr><td> 25 mg Insulin-Lösung </td><td> 4 ml </td></tr><tr><td> Selen </td><td> 100 &amp; mu; L </td></tr><tr><td> Progesteron </td><td> 100 ul </td></tr></tbody></table> Hormone Mix (100 ml insgesamt bei -20 ° C) <table border="1"><tbody><tr><td> Artikel </td><td> Volumen </td></tr><tr><td> Wasser autoklaviert </td><td> 37,5 ml </td></tr><tr><td> 10X DMEM/F12 (3:1) </td><td> 5 ml </td></tr><tr><td> 30% Glucose </td><td> 1 ml </td></tr><tr><td> 7,5% NaHCO 3 </td><td> 0,75 ml </td></tr><tr><td> 1M HEPES </td><td> 0,25 ml </td></tr><tr><td> Hormone mix </td><td> 5 ml </td></tr><tr><td> L-Glutamin </td><td> 0,5 ml </td></tr><tr><td> Penicillin-Streptomycin </td><td> 0,5 ml </td></tr></tbody></table> Serum Free Media EFH-SFM: add 10 ul EGF, 10 ul FGF und 3,66 ul Heparin FBS-SFM: 0,5 ml FBS <talich border = &quot;1&quot;&gt; <tbody><tr><td> Artikel </td><td> Volumen </td></tr><tr><td> Matrigel </td><td> 150 ul </td></tr><tr><td> SFM </td><td> 3,6 ml </td></tr></tbody></table> Matrigel Lösung Matrigel Aliquot sollte in einer Box von Eis gelegt werden und langsam auftauen über 4-5 Stunden, um eine viskose Flüssigkeit vor dem Mischen mit SFM bilden. Dadurch wird die Bildung einer glatten Schicht aus Matrigel Substrat. Wenn nicht aufgetaut langsam, wird das resultierende Substrat enthalten Klumpen von Matrigel, möglicherweise behindern Zellmigration. <table border="1"><tbody><tr><td> Artikel </td><td> Volumen oder Masse </td></tr><tr><td> UltraPure Agarose </td><td> 300 mg in 10 ml ddH 2 0 </td></tr><tr><td> SFM Hitzeinaktiviertem FBS </td><td> 8 ml 2 ml </td></tr></tbody></table> MatrigelLösung Mix 8 ml SFM mit 2 ml hitzeinaktiviertem FBS in einer 15 cc Falcon-Röhrchen. Mischung Agarose mit 10 ml ddH 2 0 in einem Erlenmeyerkolben und Hitze in einer Mikrowelle für 30 sec. in 10-sec Intervallen, wodurch die Lösung aus der Mikrowelle zu entfernen, nachdem jedes 10-Sekunden-Intervall und gründlich zu mischen. Nach dem abschließenden 10-sec Mikrowelle Zeitraum mischen die Agarose-Lösung mit der SFM / FBS-Lösung und an einem 57 ° C Wasserbad. </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

Die Entdeckung von neuralen Stamm-und Vorläuferzellen (kollektiv als neuralen Vorläuferzellen) (NPC) im erwachsenen Gehirn von Säugetieren ist an einem Körper der Forschung auf die Nutzung der multipotenten und proliferativen Eigenschaften dieser Zellen für die Entwicklung von Strategien neuroregenerative gerichtet geführt. Ein entscheidender Schritt für den Erfolg einer solchen Strategie ist die Mobilisierung von NSC Richtung einer Läsionsstelle nach Transplantation oder exogene, um die Reaktion der endogenen Vorstufen, die in der periventrikulären Region des ZNS gefunden werden verbessern. Dementsprechend ist es wichtig, die Mechanismen, die zu fördern leiten, und verbessern NPC Migration verstehen. Unsere Arbeit konzentriert sich auf die Nutzung von Gleichstrom elektrische Felder (dcEFs) zu fördern und zu direkten NPC Migration - ein Phänomen, das als Galvanotaxis bekannt. Endogenen physiologischen elektrischen Feldern funktionieren als kritische Hinweise für die Zellwanderung während der normalen Entwicklung und Wundheilung. Pharmakologische Unterbrechung dertrans-Neuralrohr Potenzial Axolotl Embryonen verursacht schweren Entwicklungsstörungen Fehlbildungen 1. Im Rahmen der Wundheilung, wird die Rate der Reparatur der verwundeten Hornhaut direkt mit der Größe der Wunde epithelialen Potential, das nach der Verletzung auftritt korreliert, als durch pharmakologische Verstärkung oder Störung dieses dcEF 2-3 gezeigt. Wir haben gezeigt, dass adulte subependymale NPCs rasche und gerichteten Migration kathodenseitigen in vitro durchlaufen, wenn sie einem von außen angelegten dcEF ausgesetzt. In diesem Protokoll beschreiben wir unser Labor die Techniken für die Schaffung eines einfachen und effektiven Galvanotaxis Assay für hochauflösende, langfristige Beobachtung der Regie Zellkörper Translokation (Migration) auf einer Ebene einzelner Zellen. Dieser Assay wäre geeignet für die Untersuchung der Mechanismen, dcEF Transduktion in zelluläre Motilität regulieren durch die Verwendung von transgenen oder Knockout-Mäuse, short interfering RNA oder spezifischen Rezeptor Agonisten / Antagonisten.

Kinematische Analyse zeigt, daß in Gegenwart von 250 mV / mm dcEF, undifferenzierte NPCs sehr gerichtet und rasche Galvanotaxis Richtung der Kathode (5A, Kinofilm 1) aufweisen. In Abwesenheit eines dcEF wird zufällige Bewegung der Zellen beobachtet (5B, Film 2). Zu dieser Feldstärke&gt; 98% von undifferenzierten NSCs für die gesamte 6-8 h, für die sie abgebildet migrieren, und da tote Zellen nicht migrieren dies legt nahe, dass sie lebensfähig bleiben während dieses Zeitraums in ...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

In diesem Protokoll zeigen wir, wie Sie benutzerdefinierte Kammern, die die Anwendung eines Gleichstrom elektrisches Feld an Zeitraffer-Aufnahmen von erwachsenen Gehirn abgeleitete neurale Vorläuferzellen Translokation während Galvanotaxis ermöglichen ermöglichen konstruieren.

A Galvanotaxis Assay für die Analyse von neuralen Vorläuferzellen Cell Migration Kinetics in einem von außen angelegten Direct Current Electric Field

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

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Research

JoVE Journal

Neuroscience

12.4K Aufrufe.  University of Toronto. In diesem Protokoll zeigen wir, wie Sie benutzerdefinierte Kammern, die die Anwendung eines Gleichstrom elektrisches Feld an Zeitraffer-Aufnahmen von erwachsenen Gehirn abgeleitete neurale Vorläuferzellen Translokation während Galvanotaxis ermöglichen ermöglichen konstruieren.

Serie: A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural-Colony Forming Zell-Assay: Ein Test zur Bona Fide neuralen Stammzellen aus neuralen Vorläuferzellen Discriminate

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Forschung

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Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Analyse der Trunk Neural Crest Cell Migration mit Hilfe eines modifizierten Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reproduzierbare Maus Ischiasnerv Crush und anschließende Bewertung der Regeneration von Whole mount Muskelanalyse

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Die Analyse der Neuralleiste Migration und Differenzierung von Cross-Spezies Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

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

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Die Neuroblast Assay: Ein Test für die Erzeugung und Anreicherung von neuronalen Vorläuferzellen aus neuralen Stammzellen differenzieren Progeny mittels Durchflusszytometrie

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolierung und Kultivierung von Zellen der Neuralleiste aus embryonalen murinen Neuralrohr

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Eine quantitative Assay für Zellmigration Murine Enteric neuralen Vorläuferzellen

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Gleichzeitige Aufnahmen der kortikalen lokales Feld Potentiale, Elektrokardiogramm, Elektromyogramm und Atemrhythmus aus einem frei beweglichen Ratte

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Weitfeld-Einzelphotonen optische Aufnahme in Gehirnscheiben mit spannungsempfindlichem Farbstoff

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

High-Content-Screening-Differenzierungs- und Reifungsanalyse von fetalen und adulten neuralen Stammzellen-abgeleiteten Oligodendrozyten-Vorläuferzellkulturen

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...