Nous tenons à remercier Marc Wozniak pour l&#39;assistance vidéo. Nous tenons également à remercier Sean Morrison à l&#39;UT Southwestern pour le protocole original de culture de cellules de rat NC. Ce travail a été soutenu Vanderbilt University Medical Center Programme de soutien académique et par des subventions du NIH (HD36720 et HD036720-11S109) et le 11GRNT7690040 AHA pour PAL, bourses doctorales de l&#39;AHA (0615209B) et le NIH (NS065604) à NAM, et l&#39;ERP a été soutenu par une subvention des NIH de formation T32HD007502.

1. Plaques Préparation <ol><li> Utiliser une technique stérile à tout moment. </li><li> Préparer la fibronectine (FN) en diluant 100 uL de plasma humain FN stock dans un volume final de 3,3 ml dans du PBS de Dulbecco (DPBS). La concentration finale est de 30 pg / ml, ce qui peut être conservé à 4 ° C pendant 1 semaine. </li><li> Recouvrez le fond de chaque puits d&#39;une culture de tissu stérile de quatre plats bien avec une solution FN et laisser reposer pendant 15 minutes. Assurez-vous que la surface est entièrement recouverte. Sensibiliser les médias pendant cette période (étapes 2 et 3). </li><li> Retirer FN solution et laisser les plaques sécher. Rincer délicatement les puits avec 500 uL de DMEM, supprimer DMEM, et ajouter 500 ul d&#39;auto-renouvellement (SR) à moyen (voir ci-dessous). Incuber à 37 ° C dans un incubateur humidifié contenant 5 pour cent de CO 2. Remplissez cette heure environ avant la dissection de sorte que les plaques seront à sec avant de l&#39;utiliser. </li></ol> 2. Préparation moyen SR <ol><li> Pour la culture de la vague et le tronc NCà partir de dix embryons (environ une portée, selon le fond génétique de la lignée de souris), de préparer 25 ml de milieu SR. Mélanger 12,5 ml de glucose à faible DMEM, 7,5 ml de milieu Neurobasal, 25 l&#39;acide rétinoïque ul (117 uM de concentration finale), et 25 pi de 2-mercaptoéthanol (50 mM de concentration finale). Mélangez bien. </li><li> Ajouter 3,75 ml extrait d&#39;embryon de poussin, 250 ul supplément sel N 2, 500 ul B27 supplément et 250 ul de pénicilline-streptomycine (1% en concentration finale). Filtrer le milieu à travers un filtre de 0,22 um. </li><li> Ajouter 10 uL stérile IGF1 (20 pg / ml de concentration finale) et 20 bFGF ul stérile (20 pg / ml de concentration finale). Mélanger par inversion. Conserver à 4 ° C. </li></ol> 3. Préparation Lavable <ol><li> Pour environ 10 embryons de préparer 50 ml de milieu de lavage. Mélanger 50 mg de BSA avec 35 ml de glucose à faible DMEM, 15 ml de milieu Neurobasal, et 500 ul de pénicilline-streptomycine (1% en concentration finale). Filtre stérile avec un 0,22Filtre um. </li></ol> 4. Préparation de la collagénase / dispase <ol><li> Ajouter 50 uL de 100 mg / ml de collagénase / dispase à 5 mL dPBS. Mélangez bien. </li><li> Seringue filtre avec un filtre de 0,2 um et ajoutez 1,5 ml dans chacun des trois puits d&#39;une plaque douze bien. Introduire à la pipette d&#39;environ 1 ml milieu de lavage dans les puits restants. Conserver la plaque entière sur la glace jusqu&#39;à ce que prêt à digérer les tissus disséqués. </li><li> Couper conseils d&#39;un embout de pipette p20 et p1000 filtre avec une lame de rasoir stérile. Coupez juste au-dessous du bord biseauté de la pointe. La coupure sera p1000 être utilisé pour transférer l&#39;embryon entier tandis que le p20 sera utilisé pour transférer des morceaux de tissu isolé. </li></ol> 5. Isoler vagal et Tube Neural Trunk à partir d&#39;embryons DPC 9.5 <ol><li> Pour les grossesses chronométrés, les barrages avec un bouchon vaginal sont considérés 0,5 dpc à midi le matin, le bouchon est observée. Sacrifice et enlever l&#39;utérus à 9,5 dpc. </li><li> Retirez la caduque de l&#39;utérus et doucement remove l&#39;embryon de la caduque. Une fois connu avec ce protocole, isoler 3-4 9.5 embryons DPC à la fois dans dPBS stériles. Utiliser une technique stérile et instruments de dissection stérilisés. Les instruments peuvent être stérilisés par autoclavage stérilisation par la chaleur, ou par incubation dans de l&#39;éthanol. </li><li> Pour antérieure vagal NC: l&#39;utilisation d&#39;aiguilles à insuline, couper le tube neural à la mi placode otique. Couper à nouveau le bord postérieur du somite 4 e. Coupez le tissu ventral du tube neural de supprimer les arcs branchiaux et le cœur. </li><li> Pour NC tronc: l&#39;utilisation d&#39;aiguilles à insuline, enlever la partie du tube neural entre somites 16-22 (ou le dernier somite si les embryons sont développemental plus tôt que l&#39;étape 22 somite). </li><li> Gardez le sac vitellin et tout tissu restant embryonnaire pour le génotypage. </li></ol> 6. Retrait de la non-neuronal ectoderme et du mésoderme <ol><li> Placez tube neural contenant des segments dans la collagénase / dispase à température ambiante pendant 10 minutess. Laver immédiatement au milieu de lavage. </li><li> Retourner tissu à dPBS stériles. Utiliser des aiguilles stériles à insuline, retirez délicatement l&#39;ectoderme non neural du tissu et de séparer les somites loin du tube neural. Les dernières parties restantes du mésoderme à partir de tissus somite peut être retiré en triturant délicatement avec une cut-down p20 pointe. Attention à ne pas endommager le tube neural. Observez attentivement la situation au cours de la trituration. </li><li> Placer le tube neural isolé par un lavage des deuxième et troisième 30 secondes de milieu de lavage. </li><li> Laver une fois dans un milieu SR, et placer le tube isolé neuronal dans le centre d&#39;un puits FN-revêtu qui a été préparé précédemment (étape 1.3). Humidifier la chambre de l&#39;hypoxie avec un plat d&#39;eau stérile. Placer la capsule dans la chambre de l&#39;hypoxie et rincer la chambre avec le gaz mixte à 3% d&#39;O 2 (utiliser un réservoir contenant un mélange de 1% de O 2, 6% de CO 2, 93% de N 2). </li><li> Toujours manipuler la chambre avec un soin extrême pour assurer l&#39;eexplants t ne sont pas perturbées et restent dans le centre du puits. </li><li> Incuber à 37 ° C. Résumé des étapes 5-6 est illustré à la figure 1. </li></ol> 7. Retrait du tube neural <ol><li> Après 24 heures d&#39;incubation, retirer le tube neural par un léger taquiner le bord du tube neural à l&#39;écart des cellules qui migrent à l&#39;aide d&#39;une aiguille d&#39;insuline stérile. Retirer et jeter le tube neural à partir du milieu en utilisant un coupe p20 stérile comme à l&#39;étape 6.2 et remplacer le milieu avec SR milieu frais (Figure 2). Ceci est plus facile à faire à l&#39;aide d&#39;un microscope inversé. </li></ol> 8. Les résultats représentatifs Après 24 heures d&#39;incubation à 37 ° C dans des conditions hypoxiques, les cellules ont migré loin NC à partir du tube neural dans une population presque pur (figure 3a). Parfois, moins de cultures idéales ne cédera pas excroissances solides. Par exemple, il est possible que, après 24 heures til aura du tube neural recroquevillé sur lui-même et le NC ne sera pas migrer loin du tube neural (figure 3b). Parfois, le tube neural ne se fixe pas sur les plaques recouvertes de fibronectine. Dans notre expérience, sous-optimale de migration NC ou des problèmes de fixation du tube neural peuvent être altérés par les conditions de normoxie ou de la concentration de la fibronectine, respectivement. L&#39;activité enzymatique de la collagénase / dispase varie légèrement selon le lot et le temps de digestion doivent être ajustés de manière appropriée, cependant, ne digèrent pas le tissu de plus de quinze minutes. Overdigestion du tube neural contenant des tissus dans la collagénase / dispase se traduira également par des excroissances déficientes. Si le tissu somite ne s&#39;enlève pas facilement à partir du tube neural après incubation dans la collagénase / dispase, le tube neural peuvent être incubés pendant plus de dix minutes. Parfois, le tube neural ne se fixe pas sur le substrat. Si tel est le cas, vérifiez l&#39;fibronecconcentration en étain et les conditions de l&#39;hypoxie. Bien que les conditions normoxiques peut être utilisé pour la culture de type sauvage NC, des conditions hypoxiques imiter de plus près l&#39;environnement in vivo 29,30. Dans notre expérience, des conditions hypoxiques est devenue critique lorsque la culture mutant NC. Par exemple, lorsque Foxd3 mutant NC ont été cultivées dans des conditions normoxiques, le tronc NC eu une excroissance de cellules fortement réduite par rapport aux contrôles. Cette disparité dans la taille excroissance a été supprimée lorsque les explants étaient cultivés dans des conditions hypoxiques (Figure 4). En outre, lorsque de type sauvage explants du tube neural ont été cultivées en normoxie, le nombre de cellules positives pour la caspase-était plus grande alors que des explants cultivés en hypoxie similaires (données non présentées). En maintenant toute la culture NC en hypoxie, les comparaisons ne peuvent plus facilement être faite entre la dynamique de contrôle et les cultures mutantes. <img src="/files/ftp_upload/4134/4134fig1.jpg" alt="Figure 1" /> <strong&gt; Figure 1. Schématique l&#39;ensemble de l&#39;isolement NC. A) Disséquer régions d&#39;intérêt à partir de l&#39;embryon. B) Le guide de tube neural dans la collagénase / dispase pendant dix minutes (ne pas dépasser quinze minutes). C) Laver en milieu de lavage. D) Disséquer l&#39;écart l&#39;ectoderme non-neural et du mésoderme. EF) Laver deux fois dans un milieu de lavage. G) Plaque en milieu auto-renouvellement. Incuber à 37 ° C dans 3% O 2 des conditions hypoxiques. <img src="/files/ftp_upload/4134/4134fig2.jpg" alt="Figure 2" /> Figure 2. Retrait par étapes du tube neural de l&#39;explant. Le tube neural doit être retirée après 24-48 heures pour éviter la contamination avec les non-NC cellules. A) Notez la frontière entre le tube neural et l&#39;excroissance NC (ligne continue). B) coupés le long du bord du tube neural avec une aiguille d&#39;insuline. C) Jeter le tube neural et de remplacer à moyen avec un milieu auto renouvelée. La ligne pointillée indique l&#39;étendue de l&#39;excroissance. Abréviation: NT, le tube neural. Figure 3. Exemples de résultats représentatifs. A) excroissance explant typique après 24 heures d&#39;incubation dans un milieu auto-renouvellement dans des conditions hypoxiques (ligne en pointillés indique l&#39;étendue de l&#39;excroissance). B) Vue agrandie d&#39;excroissance NC après 48 heures de culture. C) la culture idéale, avec un rendement moins excroissance faible. A et C ont été cultivées dans les mêmes conditions. Images de démontrer l&#39;aire de répartition naturelle de la robustesse de la culture. Ce peut être affectée par l&#39;efficacité de l&#39;isolement du tube neural, la concentration du FN, des conditions hypoxiques, et l&#39;heure dans la collagénase / dispase digestions. <img src="/files/ftp_upload/4134/4134fig4.jpg" alt="Figure 4" /> Figure 4. Dans les analyses in vitro de cultures d&#39;explants NF en normoxie par rapport hypoxie. De contrôle (type sauvage), les cellules ont migré à partir d&#39;explants NC du tube neural après 48 heures dans des conditions de culture normoxiques. En revanche, Foxd3 mutantNC avaient fortement réduit les excroissances cellulaires dans des conditions de normoxie (bords rouges marquent les contours des excroissances NC). Lorsque des explants comparables ont été cultivées dans des conditions hypoxiques, Foxd3 mutantes explants NC a augmenté comparativement à des contrôles, ce qui permet des analyses ultérieures. Remarque, ce comportement est bien corrélée avec le comportement de Foxd3 mutant NC in vivo.

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Une attention particulière devrait être accordée à l&#39;étape du développement de l&#39;embryon afin d&#39;assurer le succès de cette approche. Compter somites d&#39;embryons de souris au début est essentiel à la fois pour la phase correspondant embryons à l&#39;intérieur d&#39;une portée et de déterminer les régions correctes de tube neural pour l&#39;isolement. Une variation de un ou deux somites entre les embryons se situe dans une fourchette raisonnable du calendrier de développement, en fonction de ...

<table border="1"><tbody><tr><td> Réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> DMEM (faible taux de glucose) </td><td> Gibco / Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> Moyen 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> Entreposer dans 50 pg / ml d&#39;aliquots à -20 ° C. </td></tr><tr><td> bFGF </td><td> BD Biosciences </td><td> 354060 </td><td> Entreposer dans 25 pg / ml aliquotes à -20 ° C. </td></tr><tr><td> La fibronectine </td><td> Gibco </td><td> 33016-015 </td><td> Stocké dans 1mg/mL aliquots à -20 ° C. </td></tr><tr><td> L&#39;acide rétinoïque </td><td> Sigma </td><td> R2625 </td><td> Entreposer dans 35 pg / ml après reconstitution aliquotes dans de l&#39;éthanol à -20 ° C. </td></tr><tr><td> Le 2-mercaptoéthanol </td><td> Sigma </td><td> D-5637 </td><td></td></tr><tr><td> N 2 supplément </td><td> Gibco </td><td> 17502-048 </td><td></td></tr><tr><td> B27 supplément </td><td> Gibco </td><td> 17504-044 </td><td></td></tr><tr><td> Filtres Steriflip um 0,22 </td><td> Millipore </td><td> SCGP00525 </td><td></td></tr><tr><td> La pénicilline-streptomycine </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> 0,20 filtres pm </td><td> Corning </td><td> 431219 </td><td></td></tr><tr><td> Seringues (pour la filtration) </td><td> BD Biosciences </td><td> 301604 </td><td></td></tr&gt; <tr><td> Quatre plaques à puits </td><td> Thermo Fisher Scientific </td><td> 176740 </td><td></td></tr><tr><td> Collagénase / dispase </td><td> Roche </td><td> 269 ​​638 </td><td> Activité varie par lot. Entreposer dans 100 mg / ml d&#39;aliquots à -20 ° C. </td></tr><tr><td> Aiguilles à insuline (29 ½ Gage) </td><td> Becton Dickson </td><td> 309306 </td><td></td></tr><tr><td> Chambre hypoxie </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Analyseur d&#39;oxygène </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Forceps # 5 </td><td> Outils belle science </td><td></td><td> Pour enlever l&#39;utérus et la caduque. </td></tr><tr><td> Trypsine-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

Le feuillet embryonnaire de la crête neurale (CN) est une population progénitrices multipotentes qui provient à la face dorsale du tube neural, subit une transition épithélio-mésenchymateuse (EMT) et migre à travers l&#39;embryon, donnant lieu à divers types cellulaires 1-3. NC a également la capacité unique d&#39;influencer la différenciation et la maturation des organes cibles 4-6. Lorsque explanté in vitro, les progéniteurs NC subissent une auto-renouvellement, de migrer et se différencier en une variété de types de tissus, y compris les neurones, cellules gliales, les cellules musculaires lisses, des cartilages et des os. Multipotence NC a été décrite pour la première à partir d&#39;explants du tube neural aviaire 7-9. Dans l&#39;isolement in vitro de cellules NC facilite l&#39;étude de la dynamique de NC, y compris la prolifération, la migration et multipotence. Des travaux supplémentaires dans les systèmes aviaires et le rat ont démontré que les cellules explantées NC conservent leur potentiel de NC lorsque transplantées dans l&#39;embryon 10-13. Parce que ces propriétés inhérentes cellulaires sont conservés dans les progéniteurs NC explantés, le dosage du tube neural explant fournit une option intéressante pour l&#39;étude de la NC in vitro. Pour atteindre une meilleure compréhension de l&#39;mammifères NC, de nombreuses méthodes ont été employées pour isoler les populations NC. NC dérivés progéniteurs peuvent être cultivées à partir de post-migratoires endroits à la fois dans l&#39;embryon et l&#39;adulte pour étudier la dynamique de progéniteurs NC post-migratoires 11,14-20, mais l&#39;isolement des progéniteurs NC comme ils émigrent à partir du tube neural fournit une conservation optimale de la CN cellule potentiel et des propriétés de migration 13,21,22. Certains protocoles utilisent la fluorescence tri cellulaire (FACS) pour isoler une population enrichie en progéniteurs NC particuliers 11,13,14,17. Cependant, quand on commence avec des embryons à un stade précoce, le nombre de cellules adéquates pour les analyses sont difficiles à obtenir avec FACS, ce qui complique l&#39;isolement de début NC populations à partir d&#39;embryons individuels. Ici, nous décrivons une approche qui ne repose pas sur FACS et les résultats dans un environ 96% de la population NC pure, basée sur un journaliste Wnt1-Cre activé lignée 23. La méthode présentée ici est une adaptation de protocoles optimisés pour la culture de rat NC 11,13. Les avantages de ce protocole par rapport aux méthodes précédentes sont que 1) les cellules ne sont pas cultivés sur une couche nourricière, 2) FACS n&#39;est pas nécessaire d&#39;obtenir une population relativement pur NC, 3) prémigratoire cellules NC sont isolés et 4) les résultats sont facilement quantifiés. En outre, ce protocole peut être utilisé pour l&#39;isolement de NC de tout modèle de la souris mutante, ce qui facilite l&#39;étude des caractéristiques NC avec différentes manipulations génétiques. La limitation de cette approche est que le NC est supprimé à partir du contexte de l&#39;embryon, qui est connue pour influencer la survie, la migration et la différenciation de la 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

Isolement de la crête neurale embryonnaire à partir du tube neural facilite l&#39;utilisation de<em&gt; In vitro</em&gt; Méthodes pour étudier la migration, l&#39;auto-renouvellement, et multipotence de la crête neurale.

Isolement et culture de cellules de crêtes neurales de tube neural embryonnaire murin

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

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Neuroscience

17.1K vues.  Vanderbilt University Medical Center. Isolement de la crête neurale embryonnaire à partir du tube neural facilite l'utilisation de In vitro Méthodes pour étudier la migration, l'auto-renouvellement, et multipotence de la crête neurale.

Vidéo: 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 et la culture de neurones de la moelle épinière commissurales embryonnaires

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

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Neurosciences

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.

Production d&#39;extrait d&#39;embryon de poulet pour la culture des neurones murins Cellules souches Crest

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

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.

Fermeture du tube neural dans la culture Mouse Embryo entier

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.

Isolement et culture des cellules souches neurales Crête des follicules pileux humains

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

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.

Isolement et culture de cellules embryonnaires de rat neurones: un protocole rapide

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.

Isolement et culture de cellules endothéliales de la embryonnaire du cerveau antérieur

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

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Dérivation sans chargeur-de cellules progénitrices de la crête neurale souches pluripotentes humaines cellules

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolement et culture des adultes cellules souches neurales de la Subcallosal Zone Souris

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Isolement et Culture des embryons de souris des cellules souches neurales

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

<meta charset="utf8"></head><body>Nucléofection efficace de CSN isolées de la zone sous-ventriculaire murine

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>Pleins feux sur l’auteur : Nucléofection améliorée pour l’administration de gènes à haute efficacité dans des cultures de cellules souches neurales dérivées de la

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolement, expansion et nucléofection de cellules souches neurales de la zone sous-ventriculaire murine adulte

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...