Questo lavoro è sostenuto da NIH sovvenzione R01AR054593 e R01AR054593-S1 a Xu.

1. Preparazione delle piastre di coltura dei tessuti <ol><li> Coat ogni pozzetto con sufficiente poli-D-lisina (PDL) per coprire il fondo del pozzo. Lasciare le piastre ad asciugare nella cappa. </li><li> Dopo che i pozzi sono asciutti, risciacquare con acqua sterile, e aspirare. Lasciare le piastre ad asciugare nella cappa. </li><li> Quando asciutto, cappotto con fibronectina (che è stato disciolto in acqua BioWhittaker notte a 37 ° C ad una concentrazione di 1 mg in 6 ml). </li><li> Aggiungere mezzo NCSC [95 ml DMEM/F12, 1 ml Penn / Strep (P / S), 1 ml N2, 2 ml B27, 100 microlitri mercaptoetanolo (2ME, 50 magazzino mM), bFGF (20 ng / ml di media), IGF -1 (20 ng / ml di mezzo) e EGF (20 ng / ml di mezzo)] prima fibronectina secco nelle piastre, </li></ol> 2. Estrarre cellule del follicolo dei capelli dal cuoio capelluto umano <ol><li> Prima di iniziare la procedura di NCSCs isolanti, bisogna preparare i rispettivi medias e reagenti (vedi Tabella 1). </li><li> Fresco di un adulto cuoio capelluto umano dalle procedure di ringiovanimento del viso o feTal tessuto cuoio capelluto viene raccolto, lavato con PBS contenente penicillina-streptomicina. </li><li> La pelle viene quindi trasferito in provette da 50 ml e incubate in DMEM con dispasi (10 mg / ml) per una notte a 4 ° C. Incubazione per 2-4 ore a 37 ° C è anche efficace. Pezzi della pelle dovrebbe essere una larghezza massima di 1 cm per consentire l&#39;enzima di penetrare. </li><li> Trasferire la pelle in una piastra Petri sterile; togliere ogni capelli dalla pelle afferrando l&#39;albero dei capelli vicino alla superficie della pelle e tirando con forza e senza intoppi. I follicoli piliferi mostrano morfologia sia anagen (Figura 1A) o telogen (Figura 1B). </li><li> Incubare i frammenti isolati nel follicolo 0,05% tripsina-EDTA (Invitrogen) per 15-20 minuti a temperatura ambiente con agitazione periodicamente, aggiungere 4 ml di DMEM con 10% FBS per arrestare la reazione. L&#39;epitelio follicolare è tripsinizzate e filtrata attraverso filtro 40 micron per ottenere una cella singola sospensione contenente cellule di varie dimensioni e forma. </li><li> Girarea 200 xg per 5 minuti, il surnatante con cura, risospendere in 1 ml di PBS contenente 2% di siero (FBS). </li><li> In alternativa, follicoli piliferi pizzicate possono essere posti in coltura senza digestione con tripsina per crescere sfere capelli in situ. </li></ol> 3. Isolamento di NCSCs follicolo pilifero mediante cell sorting citometria a flusso <ol><li> Follicolo cella singola sospensione sono stati ottenuti dalla digestione con tripsina e marcate con anticorpi anti CD271 (APC-coniugato) / HNK1 (FITC-coniugato) o CD271 / integrina alfa4 (PE-coniugato) per 40 min in ghiaccio, al buio. </li><li> Centrifugare per 5 minuti a 200 xg a temperatura ambiente e aspirare il surnatante. </li><li> Risospendere le cellule in PBS contenente 2% di siero (FBS), e prima della cernita, PI viene aggiunto per escludere le cellule morte. </li><li> Effettuare selezione cellulare mediante citometria a flusso (FACS). Raccogliere CD271 +, HNK1 + cellule doppie positive o CD271 +, alfa4 integrina + cellule doppio positive (Figura 2). </li><li> Dopo sortavoce, CD271 +, HNK1 + cellule doppie positive o CD271 +, alfa4 integrina + cellule doppie positive sono coltivate in ultra-basso piastre di fissaggio nel medio NCSC </li></ol> 4. Cultura della NCSCs primarie <ol><li> Cultura delle cellule follicolari dissociate o FACS ordinati cellule in ultra-low piastre di fissaggio nel mezzo NCSC, cambiare mezzo ogni giorno. </li><li> Controllare coltura cellulare ogni giorno al microscopio. NCSCs inizierà a formare piccoli aggregati galleggianti dopo diversi giorni e ben formate sfere in 2-5 settimane in coltura in funzione dell&#39;età dei donatori (Figura 3A). Conseguenza apparirà in un paio di giorni in corrispondenza della zona rigonfiamento e ben formati sfere in situ in diverse settimane (Figura 3B) quando i follicoli piliferi intere sono coltivate in mezzo NCSC. </li></ol> 5. Ampliamento della NCSCs <ol><li> Lavare le cellule una volta con PBS. </li><li> Aggiungere pre-riscaldata (a 37 ° C, critica) accutase e incubare a 37 ° C per 5-10min con agitazione periodicamente per assicurarsi che le sfere di cellule staminali neurali dissociato in singola cella (per NCSC allegato colto, incubare le cellule con accutase a 37 ° C per 5 minuti per assicurarsi che le cellule staminali dei follicoli piliferi a schiera). </li><li> Lavare le cellule due volte e centrifugare per 5 min a 200 xg a temperatura ambiente in mezzo DMEM/F12 per rimuovere qualsiasi residuo della soluzione accutase. </li><li> Risospendere le cellule con terreno di coltura NCSC. </li><li> Per la cultura galleggiante, mettere le cellule in ultra-low piastre di fissaggio. Per la cultura attaccamento, mettere le cellule in piastre pretrattate coltura di tessuti. </li></ol> 6. Risultati rappresentativi Mezzo di coltura NCSC è sufficiente a mantenere hNCSCs in uno stato indifferenziato senza la necessità di cellule feeder. Cheratinociti non proliferano e gradualmente morì nel mezzo. Alcune piccole cellule rotonde proliferare e formare piccoli aggregati in sospensione dopo 3 a 5 giorni. Tali aggregati galleggianti lentamente aumentato in dimensioni, generati sfera tridimensionale strutture simili, che abbiamo chiamato sfere capelli (Figura 3A). Se coltivate in piastre rivestite, le NCSCs si attaccano alla superficie e non costituiscono alcun sfere. Le NCSCs allegati crescere più velocemente in sospensione. Le cellule staminali di formazione di sfere o fissati esprimono marcatori NCSCs. Quando i follicoli piliferi sono intere culture, sfere si formano alla superficie corrispondente alla regione di rigonfiamento (Figura 3B). <img alt="Figura 1" src="/files/ftp_upload/3194/3194fig1.jpg" /> Figura 1. Pizzicate anagen (A) e telogen (B) follicoli piliferi. <img alt="Figura 2" src="/files/ftp_upload/3194/3194fig2.jpg" /> Figura 2. Immagini rappresentative della analisi FACS di NCSCs pannello sinistro.: Le cellule sono gated utilizzando anticorpi anti-CD271 e anti-alphe quattro anticorpi integrine. Pannello di destra: le cellule sono gated utilizzando anticorpi anti-CD271 e anti-HNK1. <img alt="Figura 3" src="/files/ftp_upload/3194/3194fig3.jpg" /> Figura 3. Morfologia di sfere capelli (A) Morfologia di sfere galleggianti capelli, pannello di sinistra:. Bassa potenza, pannello di destra:. Alta potenza (B) Morfologia della sfera capelli in situ in corrispondenza della zona rigonfiamento, pannello di sinistra: una sfera di capelli a rigonfiamento telogen, pannello di destra: una sfera di capelli nella regione rigonfiamento di un follicolo pilifero anagen.

<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>

Non abbiamo nulla da rivelare.

I metodi di isolamento e la coltura di cellule di cui sono riproducibili e robusti. Abbiamo generato NCSCs da decine di persone in una vasta gamma di età. Anche se è meglio per elaborare il tessuto subito dopo la raccolta del tessuto, abbiamo scoperto che i tessuti del cuoio capelluto può essere immagazzinato in modo sicuro nei media sul ghiaccio per il trasporto durante la notte con un impatto minimo sulla vitalità cellulare. È importante per trattare il tessuto cuoio capelluto con ant...

<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

Follicoli dei capelli sottoposti a crescita continua e ciclo del capello è un ben controllato processo che coinvolge la proliferazione delle cellule staminali e della quiete. Rigonfiamento dei capelli è una nicchia ben caratterizzato per le cellule staminali adulte 1. Questo segmento della guaina esterna della radice contiene un certo numero di diversi tipi di cellule staminali, comprese le cellule staminali epiteliali 2, cellule staminali melanociti 3 e cresta neurale come cellule staminali 4-7. Follicoli dei capelli rappresentano una fonte accessibile e ricco per i diversi tipi di cellule staminali umane. Noi e gli altri hanno isolato le cellule staminali neurali cresta (NCSCs) di follicoli piliferi umani fetali e adulti 4,5. Queste cellule staminali umane sono etichette-cellule di sostegno e sono in grado di auto-rinnovamento attraverso la divisione cellulare asimmetrica in vitro. Esprimono immaturi marcatori di cellule neurali della cresta, ma non marcatori di differenziazione. Il nostro studio dei profili di espressione hanno dimostrato di condividere un simile pattern di espressione genica con la pelle di topo immaturo neurale crest cellule. Essi mostrano multipotenza clonale che possono dare origine a linee cellulari miogeniche, melanocitari e neuronale dopo coltura in vitro clonale singola cella. Le cellule differenziate non solo acquisire lineage-specifici marcatori, ma anche dimostrare funzioni appropriate in condizioni ex vivo. Inoltre, questi NCSCs mostrano il potenziale di differenziazione verso linee mesenchimali. Differenziati cellule neuronali possono persistere nel cervello di topo e mantenere marcatori del differenziamento neuronale. E &#39;stato dimostrato che follicolo pilifero derivato NCSCs può aiutare la ricrescita dei nervi, e migliorare la funzione motoria nei topi trapiantati con queste cellule staminali seguendo sezionare lesione spinale 8. Inoltre, i nervi periferici sono state riparate con innesti di cellule staminali 9, e l&#39;impianto di pelle cellule derivate da precursori adiacenti schiacciato nervo sciatico ha portato a rimielinizzazione 10. Pertanto, i follicolo / pelle NCSCs capelli derivati ​​hanno già mostrato risultati promettenti per rLa terapia egenerative in modelli preclinici. Riprogrammazione di cellule somatiche di staminali pluripotenti indotte (iPS), le cellule hanno mostrato un enorme potenziale per la medicina rigenerativa. Tuttavia, ci sono ancora molti problemi con le cellule iPS, in particolare l&#39;effetto a lungo termine di oncogene / virus integrazione e tumorigenicità potenziale delle cellule staminali pluripotenti non sono stati adeguatamente affrontati. Ci sono ancora molti ostacoli da superare prima che le cellule iPS possono essere utilizzati per la medicina rigenerativa. Considerando che le cellule staminali sono noti per essere sicuri e sono stati utilizzati clinicamente per molti anni, come trapianto di midollo osseo. Molti pazienti hanno già beneficiato del trattamento. Autologhe cellule staminali adulte sono cellule ancora preferite per il trapianto. Pertanto, le facilmente accessibili ed espandibile cellule staminali adulte in pelle umana / follicoli piliferi sono una fonte preziosa per la medicina rigenerativa.

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

Questo articolo presenta un protocollo robusto per l&#39;isolamento e la coltura di cellule staminali neurali cresta di follicoli dei capelli umani.

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

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

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16.8K Views.  School of Medicine, University of Pennsylvania. Questo articolo presenta un protocollo robusto per l'isolamento e la coltura di cellule staminali neurali cresta di follicoli dei capelli umani.

Video: 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.

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

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

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Neuroscienze

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.

Generazione di cellule staminali neurali da scartate tessuto fetale umano corticale

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.

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

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

Isolation and Culture of 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.

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

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

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.

Un mouse, due culture: isolamento e la coltura di Adult Neural Stem Cells dalle due zone neurogena Mice individuale

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.

Derivazione libero-Feeder della cresta neurale progenitrici Cellule pluripotenti da cellule staminali umane

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.

Isolamento e la coltura di adulti neurali cellule staminali dal Subcallosal Zona mouse

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.

Sviluppo di un protocollo di raffinata per Trans-sclerale Subretinal trapianto delle cellule epiteliali del pigmento retinico umano negli occhi di topo

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.

Isolamento e coltura di cellule staminali neurali embrionali del Mouse

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.

Isolamento, coltura e caratterizzazione di cellule primarie di Schwann, cheratinociti e fibroblasti dal prepuzio umano

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