Diese Arbeit wird durch NIH R01AR054593 und R01AR054593-S1 Xu unterstützt.

Ein. Vorbereitung der Zellkulturplatten <ol><li> Mantel jeder Vertiefung mit genügend Poly-D-Lysin (PDL), um den Boden der Vertiefung abzudecken. Lassen Sie die Platten in der Haube trocknen. </li><li> Nachdem die Brunnen trocken sind, mit sterilem Wasser spülen und absaugen. Lassen Sie die Platten in der Haube trocknen. </li><li> Nach dem Trocknen Rock mit Fibronectin (BioWhittaker, die in Wasser über Nacht bei 37 ° C bei einer Konzentration von 1 mg in 6 ml) gelöst. </li><li> Fügen NCSC Medium [95 ml DMEM/F12, 1 ml Penn / Strep (P / S), 1 ml N2, 2 ml B27, 100 ul Mercaptoethanol (2ME; 50 mM Lager) IGF, bFGF (20 ng / ml Medium), -1 (20 ng / ml Medium) und EGF (20 ng / ml Medium)] vor Fibronectin trocken in den Platten, </li></ol> 2. Auszug Haarfollikel-Zellen aus menschlichen Kopfhaut <ol><li> Vor dem Start des Verfahrens zur Isolierung von Neuralleistenstammzellen, muss man die entsprechenden Medien und Reagenzien herzustellen (siehe Tabelle 1). </li><li> Frische erwachsenen Menschen Kopfhaut ab Facelift Verfahren oder fetal Kopfhautgewebe wird gesammelt, gewaschen mit PBS, das Penicillin-Streptomycin. </li><li> Die Haut wird dann in 50 ml-Röhrchen überführt und in DMEM mit Dispase (10 mg / ml) über Nacht bei 4 ° C. Inkubation für 2-4 Stunden bei 37 ° C ist ebenfalls wirksam. Hautstücke sollte eine maximale Breite von 1 cm bis zu durchdringen für Enzym zu ermöglichen. </li><li> Übertragen Sie die Haut in eine sterilisierte Petrischale abziehen jedes Haar von der Haut durch Greifen der Haarschaft nahe der Hautoberfläche und Ziehen fest und glatt. Die Haarfollikel zeigen Morphologie der beiden Anagen (Abbildung 1A) oder Telogen (Abbildung 1B). </li><li> Inkubieren der isolierten Fragmente Follikels in 0,05% Trypsin-EDTA (Invitrogen) für 15-20 min bei Raumtemperatur unter Schütteln periodisch, 4 ml DMEM mit 10% FBS, um eine Reaktion zu stoppen. Die Follikelepithels wird trypsiniert und filtriert über 40 um Filter, um eine Einzelzellen-Suspension, die Zellen von unterschiedlicher Größe und Form zu erhalten. </li><li> Drehenbei 200 xg für 5 min, verwerfen Überstand vorsichtig in 1 ml PBS, enthaltend 2% Kälberserum (FBS) erneut zu suspendieren. </li><li> Alternativ können gezupft Haarfollikel in Kultur ohne Trypsinverdau platziert werden, um Haare Kugeln in situ wachsen. </li></ol> 3. Isolation der Haarfollikel Neuralleistenstammzellen Mit Durchflusszytometrische Cell Sorting <ol><li> Haarfollikel Einzelzell-Suspension wurden durch Trypsinverdau erhaltene und mit dem Antikörper gegen CD271 (APC-konjugiertem) / HNK1 (FITC-konjugierten) oder CD271 / alpha4-Integrin (PE-konjugierten) für 40 min auf Eis im Dunkeln. </li><li> Zentrifuge für 5 min bei 200 xg bei Raumtemperatur und saugen Sie den Überstand. </li><li> Die Zellen in PBS mit 2% Serum (FBS), und vor der Sortierung wird PI Tor aus den toten Zellen aufgenommen. </li><li> Führen Zellsortierung mittels Durchflusszytometrie (FACS). Sammle CD271 +, HNK1 + doppelt positiven Zellen oder CD271 +, alpha4 Integrin + doppelt positiven Zellen (Abbildung 2). </li><li> Nach Arttion, CD271 +, HNK1 + doppelt positiven Zellen oder CD271 +, alpha4 Integrin + doppelt positiven Zellen werden in ultra-low Befestigungsplatten im NCSC kultiviert </li></ol> 4. Culture of Primary Neuralleistenstammzellen <ol><li> Kultur die abgetrennten Follikelzellen oder FACS sortierten Zellen in ultra-low Befestigungsplatten im NCSC Medium verändern Medium jeden Tag. </li><li> Überprüfen Zellkultur jeden Tag unter dem Mikroskop. Neuralleistenstammzellen beginnt schwimmende kleine Aggregate nach mehreren Tagen und wohlgeformte Kugeln in 2-5 Wochen in der Kultur abhängig vom Alter der Spender (3A) zu bilden. Outgrowth wird in wenigen Tagen bei der Ausbuchtung Region und wohlgeformten Kugeln in situ in einigen Wochen (3B), wenn ganze Haarfollikel in der NCSC Medium kultiviert erscheinen. </li></ol> 5. Ausbau der Neuralleistenstammzellen <ol><li> Waschen der Zellen einmal mit PBS. </li><li> Fügen vorgewärmten (37 ° C, kritisch) Accutase und bei 37 ° C für 5-10min unter Schütteln periodisch sicherstellen Neuronale Stammzellen Kugeln dissoziiert in einzelne Zelle (zur Befestigung kultivierten NCSC, inkubieren Zellen mit Accutase bei 37 ° C für 5 min, um sicherzustellen Haarfollikelstammzellen abgenommen). </li><li> Waschen und zentrifugieren Zellen zweimal für 5 min bei 200 × g bei Raumtemperatur in DMEM/F12 Medium, um jeglichen verbliebenen Accutase Lösung zu entfernen. </li><li> Resuspendieren Zellen mit NCSC Kulturmedium. </li><li> Für schwimmende Kultur, setzen die Zellen in ultra-low Befestigungsplatten. Zur Befestigung Kultur, setzen die Zellen in vorbehandelten Zellkulturplatten. </li></ol> 6. Repräsentative Ergebnisse NCSC Kulturmedium genügt hNCSCs in einem undifferenzierten Zustand ohne Feeder-Zellen aufrechtzuerhalten. Keratinozyten nicht vermehren und allmählich starb im Medium. Bestimmte kleine runde Zellen vermehren sich und bilden kleine Aggregate in Suspension nach 3 bis 5 Tagen. Diese schwimmenden Aggregaten langsam erhöht in Größe erzeugten dreidimensionalen Sphäre Strukturen, die wir als Haar Kugeln (3A). Wenn in beschichteten Platten kultiviert werden die Neuralleistenstammzellen an die Oberfläche binden und bildet keine Kugeln. Die beigefügten Neuralleistenstammzellen schneller wachsen als in der Schwebe. Die Kugel-bildenden oder angebrachten Stammzellen exprimieren Neuralleistenstammzellen Marker. Wenn ganze Haarfollikel Kulturen sind, werden Kugeln an der Fläche entsprechend der Ausbuchtung Bereich (3B) ausgebildet ist. <img alt="Abbildung 1" src="/files/ftp_upload/3194/3194fig1.jpg" /> Abbildung 1. Gerupft Anagen (A) und Telogen (B) Haarfollikel. <img alt="Abbildung 2" src="/files/ftp_upload/3194/3194fig2.jpg" /> Abbildung 2. Repräsentative Bilder FACS-Analyse von Neuralleistenstammzellen Linkes Feld:. Zellen sind gated mit anti-CD271-und Anti-Alphe 4-Integrin-Antikörper. Rechts: Zellen sind gated mit anti-CD271-und Anti-HNK1 Antikörper. <img alt="Abbildung 3" src="/files/ftp_upload/3194/3194fig3.jpg" /> Abbildung 3. Morphologie der Haare Kugeln (A) Morphologie von schwimmenden Haar Kugeln, linke Tafel:. Low-Power-, rechte Tafel:. Hoher Leistung (B) Morphologie der Haare Kugel in situ bei der Ausbuchtung Region, linke Tafel: ein Haar Kugel an Telogen Ausbuchtung rechte Tafel: ein Haar Kugel an der Ausbuchtung Region eines Anagen Haarfollikel.

<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>

Wir haben nichts zu offenbaren.

Die Zell-Isolierung und Kultur beschriebenen Methoden sind reproduzierbar und robust. Wir haben Neuralleistenstammzellen aus Dutzenden von Einzelpersonen in einer breiten Altersspanne generiert. Obwohl es am besten ist, um das Gewebe direkt nach Gewebe Ernte verarbeiten, fanden wir, dass der Kopfhaut Gewebe sicher in den Medien auf Eis über Nacht den Transport werden mit minimalen Auswirkungen auf die Lebensfähigkeit der Zellen gespeichert. Es ist wichtig, die Kopfhautgewebe mit Antibiotik...

<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

Haarfollikel unterliegen lebenslanges Wachstum und Haar-Zyklus ist ein gut kontrollierter Prozess, der Stammzell-Proliferation und Ruhe. Haar Ausbuchtung ist ein gut charakterisiertes Nische für adulte Stammzellen ein. Dieses Segment der äußeren Wurzelscheide enthält eine Anzahl verschiedener Typen von Stammzellen, einschließlich epithelialer Stammzellen 2, Melanocyt Stammzellen 3 und Neuralleiste wie Stammzellen 4-7. Haarfollikel sind eine zugängliche und reiche Quelle für verschiedene Arten von menschlichen Stammzellen. Wir und andere haben Neuralleiste Stammzellen (Neuralleistenstammzellen) aus humanen fötalen und adulten Haarfollikel 4,5 isoliert. Diese menschlichen Stammzellen sind Etikett haltenden Zellen und sind in der Lage, durch Selbsterneuerung asymmetrischen Zellteilung in vitro. Sie äußern unreifen Neuralleistenzellen Marker aber nicht Differenzierungsmarker. Unsere expression profiling Studie zeigte, dass sie eine ähnliche Genexpressionsmuster Aktien mit murinen Haut unreife neurale crest Zellen. Sie weisen klonale Multipotenz, die Anlass zu myogenen, melanozytären und neuronale Zelllinien geben kann nach in vitro klonale einzigen Zellkultur. Differenzierte Zellen nicht nur zu erwerben Linie-spezifische Marker, sondern auch zeigen, entsprechende Funktionen in ex vivo-Bedingungen. Darüber hinaus zeigen diese Neuralleistenstammzellen Differenzierungspotenzial Richtung mesenchymale Linien. Differenzierten neuronalen Zellen in Gehirn der Maus bestehen bleiben und zu halten neuronalen Differenzierung Marker. Es hat sich gezeigt, dass follikelstimulierendem Haar abgeleitet Neuralleistenstammzellen können Nerv Nachwachsen helfen, und sie verbessern Motorik bei Mäusen mit dieser Stammzellen transplantiert nach Durchschneiden Rückenmarksverletzung 8. Darüber hinaus haben peripheren Nerven mit Stammzell-Transplantate 9, und der Implantation von Haut-derived Vorläuferzellen neben zerkleinert Ischiasnerven repariert wurde in Remyelinisierung 10 geführt. Deshalb haben die Haarfollikel / Haut abgeleitet Neuralleistenstammzellen bereits viel versprechende Ergebnisse für r angezeigtegenerative Therapie in präklinischen Modellen. Somatische Zellen Reprogrammierung zu induzierten pluripotenten Stammzellen (iPS-Zellen) hat ein enormes Potential für die regenerative Medizin dargestellt. Allerdings gibt es noch viele Probleme mit iPS-Zellen, insbesondere die langfristigen Auswirkungen von Onkogen / Virus Integration und potenzielle tumorigenicity von pluripotenten Stammzellen wurden nicht angemessen behandelt. Es gibt noch viele Hürden zu überwinden, bevor iPS-Zellen für die regenerative Medizin verwendet werden können. Während die adulten Stammzellen sind bekannt als sicher und sie wurden klinisch seit vielen Jahren, z. B. Knochenmarkstransplantation verwendet. Viele Patienten haben bereits von der Behandlung profitiert. Autologen adulten Stammzellen sind noch bevorzugte Zellen für die Transplantation. Daher sind die leicht zugänglich und erweiterbar adulten Stammzellen in der menschlichen Haut / Haarfollikel eine wertvolle Quelle für die regenerative Medizin.

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

Dieser Artikel stellt eine robuste Protokoll zur Isolierung und Kultivierung der Neuralleiste Stammzellen aus menschlichen Haarfollikeln.

Isolation und Kultur von Neural Crest Stem Cells aus menschlichen Haarfollikeln

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

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16.8K Aufrufe.  School of Medicine, University of Pennsylvania. Dieser Artikel stellt eine robuste Protokoll zur Isolierung und Kultivierung der Neuralleiste Stammzellen aus menschlichen Haarfollikeln.

Serie: 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.

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

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

Forschung

Neurowissenschaften

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.

Generierung von neuralen Stammzellen aus menschlichen Föten Ausrangierte Rindengewebe

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.

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

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

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

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

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

Isolierung und Kultivierung von Zellen der Neuralleiste aus embryonalen murinen Neuralrohr

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

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.

Eine Maus, zwei Kulturen: Isolierung und Kultur von adulten neuralen Stammzellen aus dem Zwei Neurogene Zonen der einzelnen Mäuse

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.

Feeder freie Ableitung von Neural Crest Vorläuferzellen aus menschlichen pluripotenten Stammzellen

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.

Isolierung und Kultur von adulten neuralen Stammzellen aus der Maus subcallosa Zone

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.

Entwicklung eines raffinierten Protokolls für Trans-skleralen subretinalen Transplantation von menschlichen retinalen Pigment Epithelzellen in Ratte Augen

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.

Isolation und Kultur der embryonalen Maus neurale Stammzellen

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.

Isolierung, Kultur und Charakterisierung von primären Schwann-Zellen, Keratinozyten und Fibroblasten aus der menschlichen Vorhaut

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