Wir möchten Marc Wozniak für Video-Hilfe anzuerkennen. Wir möchten auch Sean Morrison an der UT Southwestern anerkennen für das Original-Protokoll zur Kultivierung von Ratten-NC-Zellen. Diese Arbeit wurde Vanderbilt University Medical Center Academic Program-Support unterstützt und durch Zuschüsse der NIH (HD36720 und HD036720-11S109) und der AHA 11GRNT7690040 zu PAL, Chemiefonds Stipendien von der AHA (0615209B) und NIH (NS065604) zu NAM und ERP war unterstützt durch ein NIH Ausbildungsförderung T32HD007502.

1. Vorbereiten von Platten <ol><li> Verwenden steriler Technik zu jeder Zeit. </li><li> Planen Fibronektin (FN) durch Verdünnen von 100 ul Blutplasma FN Brühe in einem Endvolumen von 3,3 ml in Dulbeccos PBS (DPBS). Endkonzentration 30 ug / ml, und dies kann bei 4 ° C für 1 Woche aufbewahrt werden. </li><li> Titelbild Boden jeder Vertiefung einer sterilen Gewebekultur vier Well-Platte mit FN-Lösung und lassen Sie sich für 15 Minuten. Sicherstellen, dass die gesamte Oberfläche bedeckt ist. Machen Medien während dieser Zeit (die Schritte 2 und 3). </li><li> Entfernen Sie FN-Lösung und erlauben Platten zum Trocknen auf. Vorsichtig spülen Wells mit 500 ul DMEM, DMEM entfernen, und fügen Sie 500 ul der Selbsterneuerung (SR) Medium (siehe unten). Bei 37 ° C in einem befeuchteten Inkubator mit 5 Prozent CO 2. Füllen Sie dieses etwa eine Stunde vor ihrer Zerlegung, so dass die Platten werden vor Gebrauch trocknen. </li></ol> 2. Vorbereiten SR Medium <ol><li> Für die Kultur des Vagus und des Rumpfes NCaus zehn Embryonen (etwa ein Wurf, je nach genetischem Hintergrund der Mauslinie), bereiten Sie 25 ml Medium SR. Kombinieren 12,5 ml niedrige Glukose-DMEM, 7,5 ml Neurobasalmedium, 25 ul Retinsäure (117 uM Endkonzentration) und 25 ul 2-Mercaptoethanol (50 mM Endkonzentration). Gut mischen. </li><li> In 3,75 ml Hühnerembryo-Extrakt, 250 ul N 2 Salz Ergänzung, 500 ul B27 Ergänzung und 250 ul Penicillin-Streptomycin (1% Endkonzentration). Filtern Sie das Medium durch ein 0,22 um-Filter. </li><li> Fügen Sie 10 ul sterilem IGF1 (20 ug / ml Endkonzentration) und 20 ul sterilem bFGF (20 ug / ml Endkonzentration). Mischen durch Invertieren. Lagerung bei 4 ° C. </li></ol> 3. Vorbereiten Waschmedium <ol><li> Für etwa 10 Embryonen vorzubereiten 50 ml Waschmedium. Kombinieren Sie 50 mg BSA mit 35 ml niedrige Glukose-DMEM, 15 ml Neurobasalmedium, und 500 ul Penicillin-Streptomycin (1% Endkonzentration). Sterilfilter mit einem 0,22um-Filter. </li></ol> 4. Vorbereiten Kollagenase / Dispase <ol><li> Dann werden 50 ul 100 mg / ml Collagenase / Dispase bis 5 ml DPBS. Gut mischen. </li><li> Spritze mit Filter einen 0,2 um Filter und 1,5 mL in jedes der drei Vertiefungen einer zwölf Well-Platte. Pipette ca. 1 ml Wasch-mittel in den restlichen Brunnen. Bewahren gesamte Platte auf Eis, bis bereit, seziert Gewebe zu verdauen. </li><li> Schneiden Sie Tipps eines P20 und P1000 Pipettenfilter Spitze mit einer sterilen Rasierklinge. Kurz unterhalb der abgeschrägten Kante der Spitze. Die abgeschnittenen P1000 wird verwendet, um den ganzen Embryo zu übertragen, während die p20 wird verwendet, um Stücke von isoliertem Gewebe übertragen werden kann. </li></ol> 5. Isolating Vagale und Trunk Neuralrohrs aus 9,5 DPC Embryonen <ol><li> Für zeitgesteuerte Schwangerschaften, sind Dämme mit einer vaginalen Stecker 0,5 DPC mittags als der Morgen der Stecker wird beobachtet. Opfern und entfernen die Gebärmutter bei 9,5 DPC. </li><li> Entfernen Sie die Decidua von Gebärmutter und sanft rntfernen den Embryo aus der Decidua. Wenn mit diesem Protokoll erfahren, zu isolieren 3-4 9,5 dpc Embryonen zu einem Zeitpunkt in sterilem dPBS. Verwenden steriler Technik und sterilisiert Dissektionsinstrumente. Instrumente können durch Autoklavieren, Hitzesterilisation oder durch Inkubation in Ethanol sterilisiert werden. </li><li> Für Front-vagalen NC: mit Insulin Nadeln, schneiden Sie das Neuralrohr in der Mitte Ohrplakode. Schneiden Sie wieder an der hinteren Kante des 4. Somiten. Trim Gewebe ventral des Neuralrohrs, die Schlundbögen und Herz zu entfernen. </li><li> Für Stamm NC: mit Insulin Nadeln, entfernen Sie den Teil des Neuralrohrs zwischen Somiten 16-22 (oder der letzte Somiten, wenn Embryonen sind entwicklungsgeschichtlich älter als die 22 Somiten-Stadium). </li><li> Halten Sie den Dottersack und alle verbleibenden embryonalem Gewebe für die Genotypisierung. </li></ol> 6. Entfernung von nicht-neuronalen Ektoderm und Mesoderm <ol><li> Legen Neuralrohr, die Segmente in Collagenase / Dispase bei Raumtemperatur für 10 Minutens. Unmittelbar in Waschmedium zu waschen. </li><li> Zurück zur sterilen Gewebe dPBS. Mit einer sterilen Insulin-Nadeln, entfernen Sie vorsichtig die nicht-neuronalen Ektoderm aus dem Gewebe und trennen Sie die Somiten vom Neuralrohr. Die letzten Teile des Mesoderm von Somiten Gewebe kann durch Verreiben sanft mit einem Cut-down p20 Spitze entfernt werden. Seien Sie vorsichtig, um nicht zur Beschädigung des Neuralrohrs. Beachten Sie dies beim Zerreiben eng. </li><li> Platzieren Sie die isolierten neuronalen Rohr durch einen zweiten und dritten 30 Sekunden Waschgang Waschmedium. </li><li> Waschen einmal SR Medium, und legen die isolierten neuronalen Rohr in der Mitte eines FN-beschichteten, die zuvor hergestellt wurde (Schritt 1.3). Befeuchten die Hypoxie Kammer mit einem Gericht aus sterilem Wasser. Anschließend wird die Schale in die Kammer Hypoxie und spülen Sie die Kammer mit Mischgas bis 3% O 2 (verwenden Sie einen Tank mit einer Mischung aus 1% O 2, 6% CO 2, 93% N 2). </li><li> Behandeln Sie die Kammer extrem vorsichtig zu gewährleisten that Explantate sind ungestört und bleiben in der Mitte der Brunnen. </li><li> Bei 37 ° C. Zusammenfassung der Schritte 5-6 wird in 1 gezeigt. </li></ol> 7. Die Entfernung von Neuralrohrdefekten <ol><li> Nach 24 Stunden Inkubation, entfernen das Neuralrohr durch leichtes necken den Rand des Neuralrohr von den wandernden Zellen unter Verwendung einer sterilen Nadel Insulin. Entfernen und Entsorgen der Neuralrohr aus dem Medium mit einer sterilen p20 Schnitt wie in Schritt 6,2 und ersetzen Sie das Medium durch frisches Medium SR (Abbildung 2). Dies geschieht am einfachsten mit einem inversen Mikroskop. </li></ol> 8. Repräsentative Ergebnisse Nach 24 Stunden Inkubation bei 37 ° C in hypoxischen Bedingungen, NC-Zellen haben sich von dem Neuralrohr in einer nahezu reinen Population (Abbildung 3a) migriert. Manchmal wird als ideal Kulturen nicht nachgeben robust Auswüchse. Zum Beispiel ist es möglich, dass nach 24 Stunden ter Neuralrohr wird auf sich selbst zusammengerollt haben und der NC wird nicht wandern weg von der Neuralrohrdefekte (Abbildung 3b). Gelegentlich wird das Neuralrohr nicht auf die mit Fibronectin beschichteten Platten zu befestigen. Nach unserer Erfahrung können suboptimale NC Migration oder Probleme mit der Befestigung des Neuralrohrs negativ durch Normoxie Bedingungen oder Konzentration des Fibronektin betroffen sein, jeweils. Die enzymatische Aktivität der Kollagenase / Dispase variiert leicht von Batch-und Verdauung Zeit entsprechend angepasst werden müssen, jedoch nicht verdauen das Gewebe länger als 15 Minuten. Overdigestion des neuronalen Gewebes in Röhrchen mit Collagenase / Dispase auch zu einer mangelhaften Auswüchse führen. Wenn Somiten Gewebe nicht leicht von dem Neuralrohr nach Inkubation in Collagenase / Dispase entfernt wird, kann das Neuralrohr länger als zehn Minuten lang inkubiert werden. Manchmal das Neuralrohr nicht an dem Substrat. Wenn dies der Fall, überprüfen Sie die fibronecZinn-Konzentration und die Hypoxie Bedingungen. Während normoxischen Bedingungen zur Kultur Wildtyp-NC verwendet werden können, hypoxischen Bedingungen genauer nachahmen die in vivo-Umgebung 29,30. Nach unserer Erfahrung wurde hypoxischen Bedingungen kritisch, wenn die Kultivierung Mutanten NC. Zum Beispiel, wenn Foxd3 Mutante NC in normoxischen Bedingungen kultiviert wurden, hatten Stamm NC eine stark reduzierte Zelle Auswachsen im Vergleich zu Kontrollen. Dieses Missverhältnis in Auswuchs Größe wurde entfernt, wenn die Explantate in hypoxischen Bedingungen (Abbildung 4) kultiviert wurden. Darüber hinaus, wenn Wildtyp-Neuralrohr Explantaten in Normoxie kultiviert wurden, war die Anzahl der Caspase-positiven Zellen größer als die ähnlicher Explantaten in Hypoxie kultiviert (Daten nicht gezeigt). Durch die Beibehaltung aller NC-Kultur in Hypoxie, können Vergleiche zwischen einfacher Steuerung und Dynamik der mutierten Kulturen durchgeführt werden. <img src="/files/ftp_upload/4134/4134fig1.jpg" alt="1" /> <strong&gt; Abbildung 1. Insgesamt schematische von NC-Isolation. A) Dissect Regions of Interest aus dem Embryo. B) Digest Neuralrohr in Kollagenase / Dispase für 10 Minuten (nicht mehr als 15 Minuten). C) in Wasch-mittel waschen. D) Dissect weg die nicht-neuronalen Ektoderm und Mesoderm. EF) zweimal Waschen in Wasch-mittel. G) Platte in Selbsterneuerung Medium. Bei 37 ° C in 3% O 2 hypoxischen Bedingungen. <img src="/files/ftp_upload/4134/4134fig2.jpg" alt="2" /> Abbildung 2. Schrittweise Entfernung von Neuralrohrdefekten aus Explantat. Das Neuralrohr muss nach 24-48 Stunden entfernt werden, um eine Kontamination mit nicht-NC-Zellen zu verhindern. A) Man beachte die Grenze zwischen dem Neuralrohr und der NC-Auswuchs (durchgezogene Linie). B) Schneiden Sie entlang der Kante des Neuralrohrs mit einer Insulin-Nadel. C) Entsorgen Sie das Neuralrohr und ersetzen Medium durch frisches Medium selbst zu erneuern. Eine gestrichelte Linie zeigt an, inwieweit der Auswuchs. Abkürzung: NT, Neuralrohr. Abbildung 3. Beispiele für repräsentative Ergebnisse. A) Typische Explantat Auswachsen nach 24 Stunden Inkubation in Selbsterneuerung Medium in hypoxischen Bedingungen (gestrichelte Linie gibt Ausmaß der Auswuchs). B) Vergrößerte Ansicht von NC Auswuchs nach 48 Stunden in der Kultur. C) Weniger ideal Kultur mit geringer Ausbeute Auswuchs. A und C wurden unter den gleichen Bedingungen kultiviert wird. Bilder zeigen den natürlichen Verbreitungsgebieten in Kultur Robustheit. Dies kann durch die Effizienz des Neuralrohrs Isolation, Konzentration der FN, hypoxischen Bedingungen, und die Zeit in Kollagenase / Dispase Verdauung beeinträchtigt werden. <img src="/files/ftp_upload/4134/4134fig4.jpg" alt="Abbildung 4" /> Abbildung 4. In-vitro-Analysen von NC Explantatkulturen in Normoxie gegenüber Hypoxie. Control (Wildtyp) NC-Zellen von Neuralrohr-Explantaten nach 48 Stunden in normoxischen Kulturbedingungen migriert. Im Gegensatz dazu Foxd3 MutanteNC war stark Zelle Auswüchse in Normoxie (rote Konturen Marke Kanten der NC Auswüchse) reduziert. Als vergleichbar Explantate unter hypoxischen Bedingungen angebaut wurden, wuchs Foxd3 mutierten NC-Explantate vergleichsweise zu den Kontrollen, so dass spätere Analysen. Beachten Sie, dieses Verhalten auch mit dem Verhalten der Foxd3 Mutante NC in vivo korreliert.

<ol>
	<li>Le Douarin, N., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The neural crest. , (1999).</li><li>Kulesa, P. M., Gammill, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+migration:+patterns,+phases+and+signals.">Neural crest migration: patterns, phases and signals.</a> Developmental biology. 344, 566-568 (2010).</li><li>Saint-Jeannet, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neural crest induction and differentiation. , (2006).</li><li>Plank, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+and+timing+of+arrival+of+murine+neural+crest+on+pancreatic+beta+cell+development+and+maturation.">Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.</a> Developmental biology. 349, 321-330 (2011).</li><li>Nekrep, N., Wang, J., Miyatsuka, T., German, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signals+from+the+neural+crest+regulate+beta-cell+mass+in+the+pancreas.">Signals from the neural crest regulate beta-cell mass in the pancreas.</a> Development. 135, 2151-2160 (2008).</li><li>Freem, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intrinsic+innervation+of+the+lung+is+derived+from+neural+crest+cells+as+shown+by+optical+projection+tomography+in+Wnt1-Cre;YFP+reporter+mice.">The intrinsic innervation of the lung is derived from neural crest cells as shown by optical projection tomography in Wnt1-Cre;YFP reporter mice.</a> J. Anat. 217, 651-664 (2010).</li><li>Cohen, A. M., Konigsberg, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonal+approach+to+the+problem+of+neural+crest+determination.">A clonal approach to the problem of neural crest determination.</a> Developmental biology. 46, 262-280 (1975).</li><li>Sieber-Blum, M., Cohen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+quail+neural+crest+cells:+they+are+pluripotent+and+differentiate+in+vitro+in+the+absence+of+noncrest+cells.">Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in vitro in the absence of noncrest cells.</a> Developmental biology. 80, 96-106 (1980).</li><li>Baroffio, A., Dupin, E., Douarin, N. M. L. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+precursors+for+neural+and+mesectodermal+derivatives+in+the+cephalic+neural+crest.">Common precursors for neural and mesectodermal derivatives in the cephalic neural crest.</a> Development. 112, 301-305 (1991).</li><li>White, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+stem+cells+undergo+cell-intrinsic+developmental+changes+in+sensitivity+to+instructive+differentiation+signals.">Neural crest stem cells undergo cell-intrinsic developmental changes in sensitivity to instructive differentiation signals.</a> Neuron. 29, 57-71 (2001).</li><li>Morrison, S. J., White, P. M., Zock, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification,+isolation+by+flow+cytometry,+and+in+vivo+self-renewal+of+multipotent+mammalian+neural+crest+stem+cells.">Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells.</a> Cell. 96, 737-749 (1999).</li><li>Bronner-Fraser, M., Sieber-Blum, M., Cohen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+the+avian+neural+crest:+migration+and+maturation+of+mixed+neural+crest+clones+injected+into+host+chicken+embryos.">Clonal analysis of the avian neural crest: migration and maturation of mixed neural crest clones injected into host chicken embryos.</a> J. Comp. Neurol. 193, 423-434 (1980).</li><li>Stemple, D. L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+stem+cell+for+neurons+and+glia+from+the+mammalian+neural+crest.">Isolation of a stem cell for neurons and glia from the mammalian neural crest.</a> Cell. 71, 973-985 (1992).</li><li>Corpening, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+live+imaging+of+enteric+progenitors+based+on+Sox10-Histone2BVenus+transgene+expression.">Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression.</a> Genesis. 49, 599-618 (2011).</li><li>Biernaskie, J. A., McKenzie, I. A., Toma, J. G., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skin-derived+precursors+(SKPs)+and+differentiation+and+enrichment+of+their+Schwann+cell+progeny.">Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny.</a> Nat. Protoc. 1, 2803-2812 (2006).</li><li>Chung, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+property+of+postmigratory+cranial+neural+crest+cells+and+their+utility+in+alveolar+bone+regeneration+and+tooth+development.">Stem cell property of postmigratory cranial neural crest cells and their utility in alveolar bone regeneration and tooth development.</a> Stem Cells. 27, 866-877 (2009).</li><li>Biernaskie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKPs+derive+from+hair+follicle+precursors+and+exhibit+properties+of+adult+dermal+stem+cells.">SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells.</a> Cell Stem Cell. 5, 610-623 (2009).</li><li>Hagedorn, L., Suter, U., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P0+and+PMP22+mark+a+multipotent+neural+crest-derived+cell+type+that+displays+community+effects+in+response+to+TGF-beta+family+factors.">P0 and PMP22 mark a multipotent neural crest-derived cell type that displays community effects in response to TGF-beta family factors.</a> Development. , 126-3781 (1999).</li><li>Heanue, T. A., Pachnis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+and+isolation+of+enteric+nervous+system+progenitors+using+Sox2.">Prospective identification and isolation of enteric nervous system progenitors using Sox2.</a> Stem Cells. 29, 128-140 (2011).</li><li>Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+in+reduced+levels+of+oxygen+promotes+clonogenic+sympathoadrenal+differentiation+by+isolated+neural+crest+stem+cells.">Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells.</a> J. Neurosci. 20, 7370-7376 (2000).</li><li>Ito, K., Morita, T., Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+clonal+analysis+of+mouse+neural+crest+development.">In vitro clonal analysis of mouse neural crest development.</a> Developmental biology. 157, 517-525 (1993).</li><li>Etchevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+chick,+mouse+or+human+neural+crest+cells.">Primary culture of chick, mouse or human neural crest cells.</a> Nat. Protoc. 6, 1568-1577 (2011).</li><li>Mundell, N. A., Labosky, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+stem+cell+multipotency+requires+Foxd3+to+maintain+neural+potential+and+repress+mesenchymal+fates.">Neural crest stem cell multipotency requires Foxd3 to maintain neural potential and repress mesenchymal fates.</a> Development. 138, 641-652 (2011).</li><li>Gammill, L. S., Gonzalez, C., Gu, C., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+of+trunk+neural+crest+migration+requires+neuropilin+2/semaphorin+3F+signaling.">Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling.</a> Development. 133, 99-106 (2006).</li><li>Gammill, L. S., Gonzalez, C., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin+2/semaphorin+3F+signaling+is+essential+for+cranial+neural+crest+migration+and+trigeminal+ganglion+condensation.">Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation.</a> Dev. Neurobiol. 67, 47-56 (2007).</li><li>Kasemeier-Kulesa, J. C., Bradley, R., Pasquale, E. B., Lefcort, F., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eph/ephrins+and+N-cadherin+coordinate+to+control+the+pattern+of+sympathetic+ganglia.">Eph/ephrins and N-cadherin coordinate to control the pattern of sympathetic ganglia.</a> Development. 133, 4839-4847 (2006).</li><li>Osborne, N. J., Begbie, J., Chilton, J. K., Schmidt, H., Eickholt, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin/neuropilin+signaling+influences+the+positioning+of+migratory+neural+crest+cells+within+the+hindbrain+region+of+the+the+chick.">Semaphorin/neuropilin signaling influences the positioning of migratory neural crest cells within the hindbrain region of the the chick.</a> Dev. Dyn. 232, 939-949 (2005).</li><li>Schwarz, Q., Maden, C. H., Vieira, J. M., Ruhrberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin+1+signaling+guides+neural+crest+cells+to+coordinate+pathway+choice+with+cell+specification.">Neuropilin 1 signaling guides neural crest cells to coordinate pathway choice with cell specification.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 6164-6169 (2009).</li><li>Simon, M. C., Keith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxygen+availability+in+embryonic+development+and+stem+cell+function.">The role of oxygen availability in embryonic development and stem cell function.</a> Nat. Rev. Mol. Cell Biol. 9, 285-296 (2008).</li><li>Ivanovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+or+in+situ+normoxia:+The+stem+cell+paradigm.">Hypoxia or in situ normoxia: The stem cell paradigm.</a> J. Cell Physiol. 219, 271-275 (2009).</li></ol>

Wir haben nichts zu offenbaren.

Besondere Aufmerksamkeit sollte auf die Entwicklungsstufe des Embryos zu zahlen, um den Erfolg dieses Ansatzes zu gewährleisten. Zählen von Somiten frühe Mausembryonen ist sowohl für die Bühne passend Embryonen innerhalb eines Wurfes und der Bestimmung der richtigen Regionen des Neuralrohrs für die Isolierung von entscheidender Bedeutung. Eine Variation von ein oder zwei Somiten zwischen Embryonen ist innerhalb einer angemessenen Palette von der zeitlichen Entwicklung, abhängig von der Auflösung des Experiments ...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> DMEM (low Glukose) </td><td> Gibco / Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> Neurobasalmedium </td><td> Gibco </td><td> 21103 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A3912-10G </td><td></td></tr><tr><td> dPBS </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> IGF1 </td><td> BD Biosciences </td><td> 354037 </td><td> Shop in 50 mu g / ml Aliquots bei -20 ° C. </td></tr><tr><td> bFGF </td><td> BD Biosciences </td><td> 354060 </td><td> Shop in 25 ug / ml Aliquots bei -20 ° C. </td></tr><tr><td> Fibronektin </td><td> Gibco </td><td> 33016-015 </td><td> Gespeichert in 1mg/ml aliquots bei -20 ° C </td></tr><tr><td> Retinsäure </td><td> Sigma </td><td> R2625 </td><td> Shop in 35 mu g / ml Aliquots nach Rekonstitution in Ethanol bei -20 ° C </td></tr><tr><td> 2-Mercaptoethanol </td><td> Sigma </td><td> D-5637 </td><td></td></tr><tr><td> N 2 Ergänzung </td><td> Gibco </td><td> 17502-048 </td><td></td></tr><tr><td> B27 Ergänzung </td><td> Gibco </td><td> 17504-044 </td><td></td></tr><tr><td> Steriflip 0,22 um Filter </td><td> Millipore </td><td> SCGP00525 </td><td></td></tr><tr><td> Penicillin-Streptomycin </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> 0,20 um-Filter </td><td> Corning </td><td> 431219 </td><td></td></tr><tr><td> Spritzen (für die Filtration) </td><td> BD Biosciences </td><td> 301604 </td><td></td></tr&gt; <tr><td> Vier-Well-Platten </td><td> Thermo Fisher Scientific </td><td> 176740 </td><td></td></tr><tr><td> Collagenase / Dispase </td><td> Roche </td><td> 269 ​​638 </td><td> Aktivität variiert je nach Charge. Shop in 100 mg / ml Aliquots bei -20 ° C. </td></tr><tr><td> Insulin Nadeln (29 ½ Gage) </td><td> Becton Dickson </td><td> 309306 </td><td></td></tr><tr><td> Hypoxie-Kammer </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Sauerstoff-Analysator </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Pinzetten # 5 </td><td> Fine Science Tools </td><td></td><td> Zur Entfernung von Gebärmutter und Decidua. </td></tr><tr><td> Trypsin-EDTA (0,25%) </td><td> Gibco </td><td> 25200 </td><td></td></tr></tbody></table>

isolation and culture of neural crest cells from embryonic murine neural tube

Die embryonalen Neuralleiste (NC) ist ein multipotenten Vorläufer-Population, die an der dorsalen Seite des Neuralrohrs stammt, erfährt eine epitheliale zu mesenchymale Transition (EMT) und wandert während des Embryos, was zu diversen Zelltypen 1-3. NC hat auch die einzigartige Fähigkeit, die Differenzierung und Reifung von Zielorganen 6.4 zu beeinflussen. Wenn es in vitro explantiert, NC Vorläuferzellen Selbsterneuerung durchlaufen, Migration und Differenzierung in eine Vielzahl von Gewebetypen einschließlich Neuronen, Gliazellen, glatten Muskelzellen, Knorpel und Knochen. NC Multipotenz wurde zum ersten Mal aus Explantaten der Vogelgrippe Neuralrohr 7-9 beschrieben. In-vitro-Isolierung von NC-Zellen erleichtert die Untersuchung der Dynamik NC einschließlich Proliferation, Migration, und Multipotenz. Weitere Arbeiten in den Vogel-und Ratten-Systemen gezeigt, dass explantierten NC-Zellen ihre NC-Potenzial zu behalten, wenn wieder in den Embryo verpflanzt 10-13. Da diese inhärenten zellulären Eigenschaften in explantierten NC Vorfahren bewahrt werden, bietet das Neuralrohr Explantat Assay eine attraktive Option für das Studium der NC in vitro. Um ein besseres Verständnis der Säuger NC zu erreichen, wurden viele Verfahren verwendet worden, um NC-Populationen zu isolieren. NC-abgeleitete Vorläuferzellen aus der Post-wandernde Standorten können sowohl im Embryo und Erwachsenen, die Dynamik des Post-NC wandernden Vorläuferzellen 11,14-20 studieren kultiviert werden, jedoch Isolierung von NC-Vorläuferzellen, wie sie aus dem Neuralrohr auswandern sorgt für eine optimale Erhaltung der NC-Zellen-Potenzial und wandernde Eigenschaften 13,21,22. Einige Protokolle verwenden fluorescence activated cell sorting (FACS), einen NC Bevölkerung für bestimmte Vorläuferzellen 11,13,14,17 bereichert zu isolieren. Allerdings, wenn beginnend mit frühen Embryonen, sind ausreichende Zellzahlen für die Analysen nur schwer mit FACS erhalten, erschwert die Isolierung des frühen NC populations aus einzelnen Embryonen. Hier beschreiben wir einen Ansatz, der nicht auf FACS Ergebnisse angewiesen ist und in einer etwa 96% reines NC Bevölkerung auf einem Wnt1-Cre-Reporter aktivierte Linie 23 basiert. Das hier vorgestellte Verfahren ist von Protokollen für die Kultur von Ratten-NC 11,13 optimiert angepasst. Die Vorteile dieses Protokoll im Vergleich zu früheren Verfahren sind, daß 1) die Zellen nicht auf einer Feeder-Schicht, 2 gezüchtet) FACS ist nicht erforderlich, eine relativ reine NC Bevölkerung, 3 zu erhalten) premigratory NC Zellen isoliert und 4) Ergebnisse sind leicht quantifiziert. Darüber hinaus ist dieses Protokoll für die Isolierung von NC von jedem mutante Maus-Modell verwendet, die die Untersuchung der NC-Eigenschaften mit unterschiedlichen genetischen Manipulationen. Die Einschränkung dieses Ansatzes ist, dass die NC aus dem Kontext des Embryos, von denen bekannt ist, um das Überleben, Migration und Differenzierung der NC 2,24-28 zu beeinflussen, wird entfernt.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

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

Isolierung von embryonalen Neuralleiste vom Neuralrohr erleichtert die Verwendung von<em&gt; In-vitro-</em&gt; Methoden zur Untersuchung von Migration, Selbsterneuerung und Multipotenz der Neuralleiste.

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 ...

isolation-culture-neural-crest-cells-from-embryonic-murine-neural

Research

JoVE Journal

Neuroscience

17.1K Aufrufe.  Vanderbilt University Medical Center. Isolierung von embryonalen Neuralleiste vom Neuralrohr erleichtert die Verwendung von In-vitro- Methoden zur Untersuchung von Migration, Selbsterneuerung und Multipotenz der Neuralleiste.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection und Kultur kommissuralen Nervenzellen aus embryonalen Rückenmark

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Forschung

Neurowissenschaften

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

Herstellung von Hühnerembryo-Extrakt für die Kultivierung von Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Gründung Embryonale Maus Neural Stem Cell Culture Mit dem Neurosphäre Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

Physiologische, morphologische und neurochemische Charakterisierung von Neuronen Modulated durch Bewegung

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Neuralrohrschlusses in Maus Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

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

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

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

Isolation und Kultur von Neural Crest Stem Cells aus menschlichen Haarfollikeln

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

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. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolierung und Kultivierung von embryonalen Ratten-Nervenzellen: ein Quick-Protokoll

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolierung und Kultivierung von Endothelzellen aus dem embryonalen Vorderhirn

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...