Vorremmo riconoscere Marc Wozniak per l&#39;assistenza video. Vorremmo inoltre ringraziare Sean Morrison del Southwestern per il protocollo originale per la coltura di cellule di ratto NC. Questo lavoro è stato supportato Vanderbilt University Medical Support programma Academic Center e da sovvenzioni da parte del NIH (HD36720 e HD036720-11S109) e il 11GRNT7690040 AHA a PAL, borse predoctoral della AHA (0615209B) e NIH (NS065604) a NAM, e ERP è stato sostenuta da una sovvenzione del NIH di formazione T32HD007502.

1. Preparazione Piastre <ol><li> Utilizzare la tecnica sterile in ogni momento. </li><li> Preparare fibronectina (FN) diluendo 100 pl di plasma umano archivio di FN in un volume finale di 3,3 ml in PBS di Dulbecco (DPBS). Concentrazione finale è di 30 ug / ml e questo può essere conservato a 4 ° C per 1 settimana. </li><li> Coprire fondo di ciascun pozzetto di una sterile coltura tissutale quattro piastre con soluzione FN e lasciate riposare per 15 minuti. Assicurarsi che l&#39;intera superficie è coperta. Assicurarsi che il supporto durante questo periodo (punti 2 e 3). </li><li> Rimuovere FN soluzione e consentire piastre asciugare. Lavare delicatamente i pozzetti con 500 microlitri DMEM, rimuovere DMEM, e aggiungere 500 microlitri di auto-rinnovamento (SR) medio (vedi sotto). Incubare a 37 ° C in un incubatore umidificato contenente il 5 percento CO 2. Completa questo circa un&#39;ora prima della dissezione in modo che le piastre saranno asciutti prima dell&#39;uso. </li></ol> 2. Preparazione Medio SR <ol><li> Per la cultura della vagale e del tronco NCda dieci embrioni (circa una cucciolata, a seconda del background genetico della linea di mouse), preparare 25 mL di mezzo di SR. Combina 12,5 mL bassa di glucosio DMEM, 7,5 ml di mezzo Neurobasal, 25 microlitri di acido retinoico (117 uM concentrazione finale), e 25 pl 2-mercaptoetanolo (50 mM di concentrazione finale). Mescolare bene. </li><li> Aggiungere 3,75 ml di estratto di Chick Embryo, 250 microlitri N 2 supplemento di sale, 500 microlitri B27 supplemento e 250 microlitri di penicillina-streptomicina (1% concentrazione finale). Filtrare il mezzo attraverso un filtro 0,22 micron. </li><li> Aggiungere 10 L IGF1 sterile (20 ug / ml concentrazione finale) e 20 pl bFGF sterile (20 ug / ml concentrazione finale). Miscelare per inversione. Conservare a 4 ° C. </li></ol> 3. Preparazione Media Wash <ol><li> Per circa 10 embrioni preparare 50 ml di mezzo di lavaggio. Combinare 50 mg BSA con 35 mL di glucosio DMEM bassa, 15 ml di mezzo Neurobasal, e 500 pl penicillina-streptomicina (1% concentrazione finale). Filtro sterile con 0,22Filtro um. </li></ol> 4. Preparazione Collagenasi / dispasi <ol><li> Aggiungere 50 pl di 100 mg / ml di collagenasi / dispasi a 5 ml DPBS. Mescolare bene. </li><li> Siringa filtro da 0,2 um filtrare e aggiungere 1,5 ml in ciascuna delle tre pozzetti di una piastra ben dodici. Pipettare circa 1 ml di mezzo di lavaggio nei pozzetti rimanenti. Conservare intera piastra sul ghiaccio fino al momento di digerire il tessuto sezionato. </li><li> Tagliare punte di una punta P20 e P1000 pipetta filtro con una lama di rasoio sterile. Tagliare appena sotto il bordo smussato della punta. Il cut off p1000 saranno utilizzati per trasferire l&#39;embrione, mentre tutta la p20 verrà utilizzato per trasferire i pezzi di tessuto isolato. </li></ol> 5. Isolamento vagale e Tube Trunk Neural da 9,5 embrioni DPC <ol><li> Per le gravidanze temporizzati, dighe con un tappo vaginale sono considerati 0,5 dpc a mezzogiorno del mattino la spina si osserva. Sacrificio e rimuovere l&#39;utero a 9,5 DPC. </li><li> Rimuovere la decidua dall&#39;utero e delicatamente rTogliete l&#39;embrione dalla decidua. Una volta sperimentato con questo protocollo, isolare 3-4 9.5 embrioni DPC in un momento in DPBS sterili. Utilizzare la tecnica sterile e strumenti di dissezione sterilizzati. Gli strumenti possono essere sterilizzati in autoclave, sterilizzazione a calore o di incubazione in etanolo. </li><li> Per vagale anteriore NC: usando aghi da insulina, tagliare il tubo neurale alla placode metà otic. Tagliare di nuovo al margine posteriore del somite 4 °. Tagliare il tessuto ventrale del tubo neurale per rimuovere gli archi faringei e il cuore. </li><li> Per NC tronco: usando aghi da insulina, rimuovere la parte del tubo neurale tra 16-22 somiti (o l&#39;ultimo somite, se gli embrioni sono evolutivamente anteriore a quella fase di 22 somite). </li><li> Tenere il sacco vitellino e qualsiasi tessuto embrionale residuo per la genotipizzazione. </li></ol> 6. Rimozione di non-neurale ectoderma e mesoderma <ol><li> Porre tubo neurale contenente segmenti in collagenasi / dispasi a temperatura ambiente per 10 minutis. Lavare immediatamente in mezzo lavaggio. </li><li> Ritorna tessuto DPBS sterili. Utilizzo di aghi da insulina sterile, rimuovere delicatamente il non-neurale ectoderma dal tessuto e separare i somiti lontano dal tubo neurale. Le parti restanti del ultime mesoderma dal tessuto somite può essere rimosso triturando delicatamente con un cut-down p20 punta. Fare attenzione a non danneggiare il tubo neurale. Osservare attentamente la situazione durante la triturazione. </li><li> Posizionare il tubo neurale attraverso isolato un secondo e il terzo lavaggio a 30 secondo di media lavaggio. </li><li> Risciacquare una volta in mezzo SR, e posizionare il tubo isolato neurale nel centro di un FN rivestita bene che è stato preparato precedentemente (Fase 1,3). Umidificare la camera di ipossia con un piatto di acqua sterile. Porre la capsula nella camera di ipossia e lavare la camera con gas miscelato al 3% O 2 (utilizzare un serbatoio contenente una miscela di 1% O 2, 6% CO 2, 93% N 2). </li><li> Maneggiare sempre la camera con estrema attenzione per garantire that espianti sono indisturbati e rimangono nel centro dei pozzetti. </li><li> Incubare a 37 ° C. Sintesi dei passaggi 5-6 è mostrato in Figura 1. </li></ol> 7. La rimozione del tubo neurale <ol><li> Dopo 24 ore di incubazione, rimuovere il tubo neurale delicatamente stuzzicando il bordo del tubo neurale lontano dalle cellule migranti usando un ago sterile di insulina. Rimuovere e gettare il tubo neurale dal mezzo mediante taglio sterile p20 come nel passaggio 6,2 e sostituire il mezzo con mezzo fresco SR (Figura 2). Questo viene fatto più facilmente utilizzando un microscopio invertito. </li></ol> 8. Risultati rappresentativi Dopo 24 ore di incubazione a 37 ° C in condizioni di ipossia, celle NC siano trasferite dal tubo neurale in una popolazione quasi puro (Figura 3a). A volte, meno di culture, ideali non produrrà escrescenze robuste. Ad esempio, è possibile che dopo 24 ore tegli tubo neurale avrà arrotolato su se stessa e il CN non si allontanino dal tubo neurale (Figura 3b). Di tanto in tanto il tubo neurale non si attaccano alle piastre rivestite fibronectina. Nella nostra esperienza, sub-ottimale la migrazione NC o problemi con il collegamento del tubo neurale può essere influenzato negativamente da condizioni normossia o concentrazione della fibronectina, rispettivamente. L&#39;attività enzimatica della collagenasi / dispasi varia leggermente per partita e tempo di digestione deve essere opportunamente regolata, tuttavia, non digerire il tessuto più di quindici minuti. Overdigestion del tubo neurale in tessuto contenente collagenasi / dispasi comporterà anche escrescenze carenti. Se il tessuto somite non viene facilmente rimosso dal tubo neurale dopo incubazione in collagenasi / dispasi, il tubo neurale può essere incubate per più di dieci minuti. Talvolta il tubo neurale non collegare al substrato. Se questo è il caso, controllare la fibronecstagno di concentrazione e le condizioni di ipossia. Mentre condizioni di normossia può essere utilizzato per coltura di tipo selvatico NC, condizioni di ipossia imitare maggiormente l&#39;ambiente in vivo 29,30. Nella nostra esperienza, condizioni di ipossia si critica quando la coltura mutante NC. Per esempio, quando Foxd3 mutante NC sono stati coltivati ​​in condizioni di normossia, tronco NC aveva una escrescenza cella notevolmente ridotto rispetto ai controlli. Questa disparità di dimensioni escrescenza stato rimosso quando gli espianti sono stati coltivati ​​in condizioni di ipossia (Figura 4). Inoltre, quando wild-type espianti tubo neurale sono state coltivate in normossia, il numero di caspasi-cellule positive era maggiore di quella di analoghi espianti coltivati ​​in ipossia (dati non mostrati). Mantenendo tutta la cultura NC in ipossia, i confronti possono più facilmente essere fatta tra le dinamiche di controllo e culture mutanti. <img src="/files/ftp_upload/4134/4134fig1.jpg" alt="Figura 1" /> <strong&gt; Figura 1. Schematica complessiva di isolamento NC. A) Dissect regioni di interesse dall&#39;embrione. B) Digest del tubo neurale in collagenasi / dispasi per dieci minuti (non superiore a quindici minuti). C) Lavare in mezzo di lavaggio. D) Dissect via il non-neurale ectoderma e mesoderma. EF) Lavare due volte in mezzo di lavaggio. G) Piatto in media rinnovamento di noi stessi. Incubare a 37 ° C in 3% O 2 condizioni di ipossia. <img src="/files/ftp_upload/4134/4134fig2.jpg" alt="Figura 2" /> Figura 2. La rimozione graduale del tubo neurale di espianto. Il tubo neurale deve essere rimosso dopo 24-48 ore per evitare la contaminazione con cellule non-NC. A) Si noti il ​​confine tra il tubo neurale e la conseguenza NC (linea continua). B) Tagliare lungo il bordo del tubo neurale con un ago da insulina. C) Eliminare il tubo neurale e sostituire media con terreno fresco rinnovamento di noi stessi. Linea tratteggiata indica la conseguenza della misura. Abbreviazione: NT, del tubo neurale. Figura 3. Esempi rappresentativi di risultati. A) escrescenza espianto tipica dopo 24 ore di incubazione in terreno di rinnovamento di noi stessi in condizioni di ipossia (linea tratteggiata indica la conseguenza della misura). B) Visualizzazione ingrandita della crescita degli NC dopo 48 ore di coltura. C) cultura meno ideale, con bassa resa escrescenza. A e C sono state coltivate nelle stesse condizioni. Le immagini mostrano la gamma naturale robustezza cultura. Questo può essere influenzata da efficienza di isolamento tubo neurale, la concentrazione di FN, condizioni di ipossia, e il tempo in collagenasi / dispasi digestioni. <img src="/files/ftp_upload/4134/4134fig4.jpg" alt="Figura 4" /> Figura 4. Nelle analisi in vitro di culture espianti NC in normossia contro l&#39;ipossia. Di controllo (tipo selvatico), le cellule migrate NC da espianti del tubo neurale dopo 48 ore in condizioni di coltura normossia. Al contrario, Foxd3 mutanteNC si era notevolmente ridotto escrescenze cellulari in normossia (bordi rossi contorni marchio delle escrescenze NC). Quando espianti comparabili sono state coltivate in condizioni di ipossia, Foxd3 mutanti espianti NC è cresciuta relativamente ai controlli, consentendo analisi successive. Nota, questo comportamento correlava bene con il comportamento di Foxd3 mutante NC in vivo.

<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>

Non abbiamo nulla da rivelare.

Particolare attenzione dovrebbe essere prestata alla fase di sviluppo dell&#39;embrione per assicurare il successo di questo approccio. Contando somiti di embrioni di topo primi è fondamentale sia per la fase di adattamento embrioni all&#39;interno di una cucciolata e la determinazione delle regioni corrette di tubo neurale per l&#39;isolamento. Una variazione di uno o due somiti tra embrioni è entro una gamma ragionevole di temporizzazione di sviluppo, a seconda della risoluzione dell&#39;esperimento condotto. Un emb...

<table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> DMEM (glucosio basso) </td><td> Gibco / Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> Media Neurobasal </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> Conservare in 50 ug / mL aliquote a -20 ° C. </td></tr><tr><td> bFGF </td><td> BD Biosciences </td><td> 354060 </td><td> Conservare in 25 ug / ml aliquote a -20 ° C. </td></tr><tr><td> Fibronectina </td><td> Gibco </td><td> 33016-015 </td><td> Conservata nella 1mg/mL aliquots a -20 ° C. </td></tr><tr><td> L&#39;acido retinoico </td><td> Sigma </td><td> R2625 </td><td> Conservare a 35 mcg / ml aliquote dopo ricostituzione in etanolo a -20 ° C. </td></tr><tr><td> 2-mercaptoetanolo </td><td> Sigma </td><td> D-5637 </td><td></td></tr><tr><td> N 2 integratore </td><td> Gibco </td><td> 17502-048 </td><td></td></tr><tr><td> B27 supplemento </td><td> Gibco </td><td> 17504-044 </td><td></td></tr><tr><td> Steriflip 0,22 micron filtri </td><td> Millipore </td><td> SCGP00525 </td><td></td></tr><tr><td> Penicillina-streptomicina </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> 0,20 micron filtri </td><td> Corning </td><td> 431219 </td><td></td></tr><tr><td> Siringhe (per filtrazione) </td><td> BD Biosciences </td><td> 301604 </td><td></td></tr&gt; <tr><td> Quattro pozzetti </td><td> Thermo Fisher Scientific </td><td> 176740 </td><td></td></tr><tr><td> Collagenasi / dispasi </td><td> Roche </td><td> 269 ​​638 </td><td> Attività varia a seconda del lotto. Conservare in 100 mg / mL aliquote a -20 ° C. </td></tr><tr><td> Insulina aghi (Calibro 29 ½) </td><td> Becton Dickson </td><td> 309306 </td><td></td></tr><tr><td> Camera di ipossia </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Oxygen Analyzer </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Pinza # 5 </td><td> Strumenti di Scienze Belle </td><td></td><td> Per la rimozione di utero e decidua. </td></tr><tr><td> Tripsina-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

Il embrionale cresta neurale (NC) è una popolazione di cellule progenitrici multipotenti che si origina nel lato dorsale del tubo neurale, subisce un epitelio di transizione mesenchimale (EMT) e migra in tutta l&#39;embrione, dando luogo a diversi tipi di cellule 1-3. NC ha anche la capacità unica di influenzare la differenziazione e la maturazione degli organi bersaglio 4-6. Quando espiantato in vitro, progenitori NC sottoposti a self-renewal, migrare e differenziarsi in una varietà di tipi di tessuto compresi i neuroni, glia, cellule muscolari lisce, cartilagine e osso. Multipotenza NC è stata descritta da espianti del tubo neurale aviaria 7-9. In isolamento in vitro di cellule NC facilita lo studio delle dinamiche NC, compresa la proliferazione, la migrazione e multipotenza. Ulteriori attività nei sistemi di aviaria e nel ratto hanno dimostrato che le cellule espiantati NC mantengono il loro potenziale NC, quando trapiantati in embrione 10-13. Poiché queste proprietà intrinseche cellulari sono conservati in espiantati progenitori NC, il saggio di espianto del tubo neurale fornisce un&#39;opzione interessante per lo studio della NC in vitro. Per ottenere una migliore comprensione del mammifero NC, molti metodi sono stati impiegati per isolare popolazioni NC. NC-derivati ​​progenitori può essere coltivato da posizioni post-migratori, sia l&#39;embrione e adulti per studiare la dinamica di post-migratori progenitori NC 11,14-20, tuttavia l&#39;isolamento di cellule progenitrici NC in quanto emigrano dal tubo neurale fornisce la migliore conservazione dei NC potenzialità e le proprietà delle cellule migratorie 13,21,22. Alcuni protocolli impiegano fluorescenza selezione delle cellule attivate (FACS) per isolare una popolazione NC arricchito per progenitori particolari 11,13,14,17. Tuttavia, quando a partire da embrioni in fase iniziale, il numero di cellule sufficienti per le analisi sono difficili da ottenere con FACS, complicando l&#39;isolamento dei primi NC populations da embrioni individuali. Qui, descriviamo un approccio che non si basa su FACS ed i risultati in una popolazione di circa il 96% NC puro basato su un Wnt1 Cre-giornalista attivato lignaggio 23. Il metodo presentato qui è l&#39;adattamento di protocolli ottimizzati per la cultura di ratto NC 11,13. I vantaggi di questo protocollo rispetto ai precedenti metodi che sono 1) le cellule non sono cresciute su uno strato alimentatore, 2) FACS non è necessaria per ottenere una popolazione relativamente puro NC, 3) NC premigratory cellule vengono isolate e 4) risultati sono facilmente quantificato. Inoltre, questo protocollo può essere utilizzato per l&#39;isolamento di NC da qualsiasi modello di topo mutante, facilitando lo studio delle caratteristiche NC con diverse manipolazioni genetiche. Il limite di questo approccio è che il NC viene rimosso dal contesto dell&#39;embrione, che è noto per influenzare la, migrazione sopravvivenza e la differenziazione del NC 2,24-28.

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

Isolamento di embrionale cresta neurale dal tubo neurale facilita l&#39;uso di<em&gt; In vitro</em&gt; Metodi per lo studio della migrazione, self-renewal, e multipotenza di cresta neurale.

Isolamento e la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

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 Views.  Vanderbilt University Medical Center. Isolamento di embrionale cresta neurale dal tubo neurale facilita l'uso di In vitro Metodi per lo studio della migrazione, self-renewal, e multipotenza di cresta neurale.

Video: 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.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

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

Ricerca

Neuroscienze

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.

Produzione di estratto di Chick dell&#39;embrione per la coltivazione delle cellule della cresta neurale Murine staminali

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.

Stabilire embrionali di topo Neural Stem Cell Culture del saggio di neurosfere

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.

Fisiologici, caratterizzazione morfologica e neurochimica dei neuroni modulati Movimenti

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.

La chiusura del tubo neurale in Cultura embrione di topo intero

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.

L&#39;isolamento e la coltura di cellule staminali neurali Crest di follicoli dei capelli umani

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.

Riproducibile nervo sciatico Crush mouse e successiva valutazione da parte della Rigenerazione intera analisi Muscle Monte

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.

Analisi della migrazione cresta neurale e la differenziazione delle specie da Cross-trapianto

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.

Isolamento e la coltura di cellule embrionali neuronali di ratto: un protocollo veloce

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.

Isolamento e coltura di cellule endoteliali dal embrionali Forebrain

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