Ce travail est soutenu par le NIH subvention R01AR054593 et ​​R01AR054593-S1 Xu.

1. Préparation des plaques de culture tissulaire <ol><li> Manteau chaque puits avec suffisamment de Poly-D-Lysine (PDL) pour couvrir le fond du puits. Laisser les plaques à sécher dans la hotte. </li><li> Après les puits sont à sec, rincez avec de l&#39;eau stérile et aspirer. Laisser les plaques à sécher dans la hotte. </li><li> Une fois sec, le manteau avec de la fibronectine (qui a été dissous dans de l&#39;eau Biowhittaker une nuit à 37 ° C, à une concentration de 1 mg dans 6 ml). </li><li> Ajouter à moyen NCSC [95 ml DMEM/F12, 1 ml Penn / Strep (P / S), 1 ml N2, 2 ml B27, 100 ul mercaptoéthanol (2ME, 50 mm stock), le bFGF (20 ng / ml de milieu), l&#39;IGF -1 (20 ng / ml de milieu) et l&#39;EGF (20 ng / ml de milieu)] avant la fibronectine sec dans les assiettes, </li></ol> 2. Extrait cellules du follicule pileux du cuir chevelu de l&#39;homme <ol><li> Avant de commencer la procédure d&#39;isolement SNCLC, il faut préparer les médias respectifs et des réactifs (voir tableau 1). </li><li> Frais peau d&#39;un adulte cuir chevelu humain des procédures lifting ou feles tissus du cuir chevelu tal est recueilli, lavé avec du PBS contenant de la pénicilline-streptomycine. </li><li> La peau est ensuite transféré dans des tubes de 50 ml et incubées dans du DMEM avec Dispase (10 mg / ml) pendant une nuit à 4 ° C. Incubation pendant 2-4 heures à 37 ° C est également efficace. Morceaux de peau doit être d&#39;une largeur maximale de 1 cm pour permettre une enzyme de pénétrer. </li><li> Transférer la peau dans un plat de Pétri stérilisées; arracher chaque poil de la peau en saisissant la tige du cheveu près de la surface de la peau et en tirant fermement et en douceur. Les follicules pileux présenter une morphologie soit anagène (figure 1A) ou télogène (figure 1B). </li><li> Incuber les fragments follicule isolés dans 0,05% de trypsine-EDTA (Invitrogen) pendant 15-20 min à température ambiante sous agitation périodique, ajouter 4 ml de DMEM avec 10% de FBS pour arrêter la réaction. L&#39;épithélium folliculaire est la trypsine et filtré à travers 40 um filtre pour obtenir une suspension à cellule unique contenant des cellules de taille variable et de forme. </li><li> Tournerà 200 xg pendant 5 min, éliminer le surnageant avec précaution, remettre en suspension dans 1 ml de PBS contenant 2% de sérum (FBS). </li><li> En variante, les follicules pileux épilé peuvent être mises en culture sans digestion par la trypsine à croître sphères cheveux in situ. </li></ol> 3. Isolement du SNCLC follicule pileux Utilisation Cell Sorting cytométrie en flux <ol><li> Follicule pileux suspension à cellule unique sont obtenus par digestion par la trypsine et étiquetées avec des anticorps contre CD271 (APC-conjugué) / HNK1 (conjugué au FITC) ou CD271 / alpha4 intégrine (PE-conjugué) pendant 40 minutes sur de la glace dans l&#39;obscurité. </li><li> Centrifuger pendant 5 min à 200 xg à température ambiante et aspirer le surnageant. </li><li> Resuspendre les cellules dans du PBS contenant 2% de sérum (FBS), et avant le tri, le PI est ajoutée à la porte les cellules mortes. </li><li> Effectuer le tri cellulaire par cytométrie en flux (FACS). Recueillir CD271 +, + HNK1 cellules doublement positives ou CD271 +, alpha4 intégrine + cellules doublement positives (figure 2). </li><li> Après sortetion, CD271 +, + HNK1 cellules doublement positives ou CD271 +, alpha4 intégrines + doubles cellules positives sont mises en culture dans des plaques de fixation ultra-faible à moyen NCSC </li></ol> 4. Culture du SNCLC primaires <ol><li> Culture des cellules folliculaires dissociées ou FACS des cellules triées dans des plaques de fixation ultra-faible dans le milieu NCSC, changer moyenne chaque jour. </li><li> Vérifiez tous les jours de culture cellulaire au microscope. SNCLC va commencer à former de petits agrégats flottants après plusieurs jours et bien formés de sphères dans 2-5 semaines dans la culture en fonction de l&#39;âge des donateurs (figure 3A). Excroissance apparaîtra dans quelques jours à la région renflement et bien formés in situ dans les sphères plusieurs semaines (figure 3B) lorsque les follicules pileux entiers sont cultivées dans le milieu NCSC. </li></ol> 5. Expansion du SNCLC <ol><li> Laver les cellules une fois avec du PBS. </li><li> Ajouter préchauffé (à 37 ° C, critique) Accutase et incuber à 37 ° C pendant 5-10min en agitant périodiquement afin de s&#39;assurer que les neurones sphères de cellules souches dissocié dans la cellule unique (NCSC attachement culture, les cellules incuber avec Accutase à 37 ° C pendant 5 minutes pour s&#39;assurer que les cellules souches du follicule pileux détaché). </li><li> Lavez et centrifuger les cellules deux fois pendant 5 min à 200 xg à température ambiante dans du milieu DMEM/F12 pour enlever toute solution restante Accutase. </li><li> Re-suspendre les cellules du milieu de culture NCSC. </li><li> Pour la culture flottante, mettre les cellules dans des plaques de fixation ultra-faible. Pour la culture attachement, mettre les cellules dans des plaques de culture de tissus prétraités. </li></ol> 6. Les résultats représentatifs NCSC milieu de culture est suffisante pour maintenir hNCSCs dans un état indifférencié sans avoir besoin de cellules nourricières. Kératinocytes prolifèrent et ne meurt peu à peu dans le milieu. Certaines petites cellules rondes vont proliférer et former de petits agrégats en suspension après 3 à 5 jours. Ces agrégats flottants lentement i augmentétaille n, générés en trois dimensions des structures de type sphère, que nous avons appelé les sphères cheveux (figure 3A). Si cultivées dans des plaques de revêtement, le SNCLC se fixer à la surface et ne forme pas de sphères. Le SNCLC jointes croître plus vite que dans la suspension. Les cellules souches sphère de formation ou attachés expriment des marqueurs SNCLC. Lorsque les follicules pileux entiers sont des cultures, les sphères sont formées au niveau de la zone correspondant à la région en boucle (figure 3B). <img alt="Figure 1" src="/files/ftp_upload/3194/3194fig1.jpg" /> Figure 1. Plumés follicules anagènes (A) et télogène cheveux (B). <img alt="Figure 2" src="/files/ftp_upload/3194/3194fig2.jpg" /> Figure 2. Des images représentatives de l&#39;analyse par FACS de SNCLC panneau de gauche: les cellules sont déclenchés. Utilisant des anticorps anti-CD271 et anti-Alphe 4 anticorps d&#39;intégrine. Panneau de droite: les cellules sont déclenchés en utilisant des anticorps anti-CD271 et anti-HNK1. <img alt="Figure 3" src="/files/ftp_upload/3194/3194fig3.jpg" /> Figure 3. Morphologie des sphères cheveux (A) Morphologie des sphères flottantes cheveux, panneau de gauche:. Faible puissance, panneau de droite:. Haute puissance (B) Morphologie de la sphère cheveux in situ dans la région renflement, panneau de gauche: une sphère cheveux au télogène renflement, panneau de droite: une sphère de cheveux à la région renflement d&#39;un follicule pileux anagène.

<ol>
	<li>Cotsarelis, G., Cheng, S. Z., Dong, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Existence+of+slow-cycling+limbal+epithelial+basal+cells+that+can+be+preferentially+stimulated+to+proliferate:+implications+on+epithelial+stem+cells.">Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.</a> Cell. 57, 201-209 (1989).</li><li>Cotsarelis, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-retaining+cells+reside+in+the+bulge+area+of+pilosebaceous+unit:+implications+for+follicular+stem+cells,+hair+cycle,+and+skin+carcinogenesis.">Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.</a> Cell. 61, 1329-1337 (1990).</li><li>Tanimura, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+stem+cells+provide+a+functional+niche+for+melanocyte+stem+cells.">Hair follicle stem cells provide a functional niche for melanocyte stem cells.</a> Cell Stem Cell. 8, 177-187 (2011).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+novel+population+of+multipotent+adult+stem+cells+from+human+hair+follicles.">Isolation of a novel population of multipotent adult stem cells from human hair follicles.</a> Am. J. Pathol. 168, 1879-1888 (2006).</li><li>Yu, H., Kumar, S. M., Kossenkov, A. V., Showe, L., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+with+neural+crest+characteristics+derived+from+the+bulge+region+of+cultured+human+hair+follicles.">Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.</a> J. Invest. Dermatol. 130, 1227-1236 (2010).</li><li>Wong, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest-derived+cells+with+stem+cell+features+can+be+traced+back+to+multiple+lineages+in+the+adult+skin.">Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin.</a> J. Cell Biol. 175, 1005-1015 (2006).</li><li>Sieber-Blum, M., Grim, M., Hu, Y. F., Szeder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+neural+crest+stem+cells+in+the+adult+hair+follicle.">Pluripotent neural crest stem cells in the adult hair follicle.</a> Dev. Dyn. 231, 258-269 (2004).</li><li>Amoh, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implanted+hair+follicle+stem+cells+form+Schwann+cells+that+support+repair+of+severed+peripheral+nerves.">Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 17734-17738 (2005).</li><li>Koliatsos, V. E., Xu, L., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell+grafts+as+therapies+for+motor+neuron+disease.">Human stem cell grafts as therapies for motor neuron disease.</a> Expert Opin. Biol. Ther. 8, 137-141 (2008).</li><li>McKenzie, I. A., Biernaskie, J., Toma, J. G., Midha, R., 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=Skin-derived+precursors+generate+myelinating+Schwann+cells+for+the+injured+and+dysmyelinated+nervous+system.">Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system.</a> J. Neurosci. 26, 6651-6660 (2006).</li><li>LeDouarin, N. M., 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>Tumbar, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+epithelial+stem+cell+niche+in+skin.">Defining the epithelial stem cell niche in skin.</a> Science. 303, 359-363 (2004).</li><li>Roh, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-potentiality+of+a+new+immortalized+epithelial+stem+cell+line+derived+from+human+hair+follicles.">Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles.</a> In Vitro Cell Dev. Biol. Anim. 44, 236-244 (2008).</li></ol>

Nous n&#39;avons rien à déclarer.

Les méthodes d&#39;isolement et de culture de cellules décrites sont reproductibles et robuste. Nous avons généré SNCLC parmi des dizaines de personnes à travers une large tranche d&#39;âge. Bien qu&#39;il soit préférable de traiter le tissu juste après la récolte des tissus, nous avons constaté que les tissus du cuir chevelu peuvent être stockés en toute sécurité dans les médias sur la glace pour le transport du jour au lendemain avec un impact minimal sur la viabilité cellulaire. <p class="jove_c...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11965-092</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>Invitrogen</td>
 <td>11330-32</td>
 </tr>
 <tr>
 <td>Heat-inactivated FBS</td>
 <td>Hyclone</td>
 <td>SH30071.03</td>
 </tr>
 <tr>
 <td>B27 supplement</td>
 <td>Invitrogen</td>
 <td>17504044</td>
 </tr>
 <tr>
 <td>N2 supplement</td>
 <td>Invitrogen</td>
 <td>17502048</td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>Invitrogen</td>
 <td>PHG0026</td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>R&amp;D system</td>
 <td>236-EG-01M</td>
 </tr>
 <tr>
 <td>IGF-I</td>
 <td>R&amp;D system</td>
 <td>291-G1-050</td>
 </tr>
 <tr>
 <td>0.05% Trypsin/EDTA</td>
 <td>Invitrogen</td>
 <td>25300-054</td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>Invitrogen</td>
 <td>17105041</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15070063</td>
 </tr>
 </tbody> 
</table>
Table 1.

isolation and culture of neural crest stem cells from human hair follicles

Les follicules pileux subissent une croissance continue et le cycle de cheveux est un processus bien contrôlé impliquant la prolifération des cellules souches et la quiétude. Renflement de cheveux est un créneau bien caractérisé pour les cellules souches adultes 1. Ce segment de la gaine épithéliale externe contient un certain nombre de différents types de cellules souches, notamment les cellules souches épithéliales 2, les cellules souches de mélanocytes 3 ​​et crête neurale comme les cellules souches 4-7. Les follicules pileux sont une source accessible et riche pour les différents types de cellules souches humaines. Nous et d&#39;autres avons isolé des cellules souches neurales de crête (SNCLC) de follicules pileux humains fœtaux et adultes 4,5. Ces cellules souches humaines sont des cellules de retenue de l&#39;étiquette et qui sont capables de s&#39;auto-renouveler par division cellulaire asymétrique in vitro. Ils expriment des marqueurs de cellules neurales immatures mais pas de crête marqueurs de différenciation. Notre étude a montré que le profil d&#39;expression qu&#39;ils partagent le même profil d&#39;expression génique similaire avec murin peau immature création de neuronescellules st. Ils présentent multipotence clonale qui peuvent donner lieu à des lignées de cellules myogéniques, mélanocytaire et neuronale après in vitro de culture cellulaire clonale unique. Les cellules différenciées non seulement acquérir la lignée des marqueurs spécifiques, mais aussi la démonstration de fonctions appropriées dans des conditions ex vivo. En outre, ces SNCLC montrer le potentiel de différenciation vers lignées mésenchymateuses. Différenciées des cellules neuronales peuvent persister dans le cerveau de souris et de conserver les marqueurs de différenciation neuronale. Il a été montré que les dérivés SNCLC follicule pileux peut aider à la repousse nerveuse, et ils améliorent la fonction motrice chez les souris transplantées avec des cellules souches à la suite de ces lésions de la moelle épinière sectionnant 8. En outre, les nerfs périphériques ont été réparés avec des greffes de cellules souches 9, et l&#39;implantation de cellules précurseurs provenant de la peau adjacente à écraser les nerfs sciatiques a abouti à la remyélinisation 10. Par conséquent, les follicules pileux de la peau / SNCLC dérivés ont déjà montré des résultats prometteurs pour rthérapie egenerative dans des modèles précliniques. Reprogrammation de cellules somatiques à des souches pluripotentes (iPS) a montré un énorme potentiel pour la médecine régénérative. Cependant, il ya encore beaucoup de problèmes avec les cellules iPS, en particulier l&#39;effet à long terme de l&#39;oncogène / virus intégration et tumorigénicité potentielle de cellules souches pluripotentes n&#39;ont pas été traitées de manière adéquate. Il existe encore de nombreux obstacles à surmonter avant que les cellules iPS peuvent être utilisés pour la médecine régénérative. Alors que les cellules souches adultes sont connus pour être en sécurité et ils ont été utilisés en clinique depuis de nombreuses années, comme la greffe de moelle osseuse. Beaucoup de patients ont déjà bénéficié de ce traitement. Autologues de cellules souches adultes sont toujours les cellules préférées pour la transplantation. Par conséquent, les cellules facilement accessibles et extensible souches adultes dans la peau de l&#39;homme / les follicules pileux sont une source précieuse pour la médecine régénérative.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles at JoVE.com

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

Cet article présente un protocole robuste pour l&#39;isolement et la culture des cellules souches neurales de crête des follicules pileux humains.

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 ...

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16.8K vues.  School of Medicine, University of Pennsylvania. Cet article présente un protocole robuste pour l'isolement et la culture des cellules souches neurales de crête des follicules pileux humains.

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

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 ...

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Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Génération de cellules souches neurales à partir de tissus humains mis au rebut corticale fœtale

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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 Neural Crest Cells from Embryonic Murine Neural Tube

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

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

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 ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Une souris, deux cultures: Isolement et culture des cellules souches adultes de neurones à partir des deux Neurogenic Zones de souris individuelle

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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 ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Élaboration d’un protocole raffiné pour Trans-sclérale Transplantation sous-rétinienne des cellules épithéliales humaines pigmentaire dans les yeux de Rat

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

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 ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

Isolement, culture et caractérisation des cellules de Schwann primaires, des kératinocytes et des fibroblastes du prépuce humain

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...