Este trabajo es apoyado por el NIH subvención R01AR054593 y R01AR054593-S1 a Xu.

1. Preparación de placas de cultivo tisular <ol><li> Escudo cada pocillo con suficiente poli-D-lisina (PDL) para cubrir el fondo del pozo. Dejar que las placas secar en la campana. </li><li> Después de que los pozos están secos, enjuague con agua estéril y aspirar. Dejar que las placas secar en la campana. </li><li> Cuando capa seca, con fibronectina (que se disolvió en agua BioWhittaker noche a 37 º C a una concentración de 1 mg en 6 ml). </li><li> Añadir medio NCSC [95 ml de DMEM/F12, 1 ml Penn / Strep (P / S), 1 ml N2, 2 ml B27, 100 l mercaptoetanol (2ME; 50 mM de existencias), bFGF (20 ng / ml de medio), IGF -1 (20 ng / ml de medio) y EGF (20 ng / ml de medio)] antes de fibronectina seco en las placas, </li></ol> 2. Extraer células del folículo piloso del cuero cabelludo humano <ol><li> Antes de iniciar el procedimiento de CTCN aislamiento, es necesario preparar las medias respectivas y reactivos (véase la Tabla 1). </li><li> Fresh piel de un adulto humano cuero cabelludo de los procedimientos de cirugía estética o FeTal tejido del cuero cabelludo se recogió, se lavó con PBS que contenía penicilina-estreptomicina. </li><li> La piel entonces se transfiere en tubos de 50 ml y se incubaron en DMEM con Dispasa (10 mg / ml) durante la noche a 4 ° C. La incubación durante 2-4 horas a 37 ° C es también eficaz. Trozos de piel debe ser de una anchura máxima de 1 cm para permitir la enzima de penetrar. </li><li> Transferencia de la piel en una placa de Petri esterilizada; salga de cada pelo de la piel, sujetando el eje del cabello cerca de la superficie de la piel y tirando firmemente y sin problemas. Los folículos pilosos muestran la morfología de cualquiera anágena (Figura 1A) o telógena (Figura 1B). </li><li> Incubar los fragmentos folículo aislado en 0,05% de tripsina-EDTA (Invitrogen) durante 15-20 minutos a temperatura ambiente con agitación periódica, añadir 4 ml de DMEM con FBS al 10% para detener la reacción. El epitelio folicular se tripsinizaron y se filtró a través de filtro de 40 micras para obtener una suspensión de una sola célula que contiene células de diferente tamaño y forma. </li><li> Girara 200 xg durante 5 min, eliminar el sobrenadante cuidadosamente, volver a suspender en 1 ml de PBS que contenía 2% de suero (FBS). </li><li> Alternativamente, desplumados folículos pilosos pueden ser colocados en cultivo sin digestión con tripsina para crecer esferas de pelo in situ. </li></ol> 3. Aislamiento de CTCN folículo piloso Uso de Ordenación Celda de flujo citométrico <ol><li> Único folículo piloso suspensión de células se obtuvieron por digestión con tripsina y se marcaron con anticuerpos contra CD271 (APC conjugado) / HNK1 (conjugado a FITC) o CD271 / integrina alfa4 (conjugado con PE) durante 40 min en hielo en la oscuridad. </li><li> Centrifugar durante 5 min a 200 xg, a temperatura ambiente y aspirar el sobrenadante. </li><li> Resuspender las células en PBS que contenía 2% de suero (FBS), y antes de la clasificación, PI se añade a la puerta de las células muertas. </li><li> Realizar la clasificación de células por citometría de flujo (FACS). Recoger CD271 +, + HNK1 células doble positivas o CD271 +, integrina alfa 4 + células doble positivas (Figura 2). </li><li> Después de claseción, CD271 +, + HNK1 células doble positivas o CD271 +, la integrina alfa 4 + células positivas dobles se cultivaron en placas de fijación ultra-bajos en el medio NCSC </li></ol> 4. Cultura de CTCN Primaria <ol><li> Cultivar las células foliculares disociadas o FACS ordenados células en placas de fijación ultra-bajos en el medio NCSC, cambie medio todos los días. </li><li> Compruebe cultivo celular todos los días bajo el microscopio. CTCN se empieza a formar pequeños agregados flotantes después de varios días y bien formadas esferas en 2-5 semanas en el cultivo dependiendo de la edad de los donantes (Figura 3A). Crecimiento Externo aparecerá en unos días en la región del bulbo y bien formados esferas in situ en varias semanas (Figura 3B) cuando los folículos pilosos se enteras cultivadas en el medio NCSC. </li></ol> 5. La expansión de CTCN <ol><li> Lavar las células una vez con PBS. </li><li> Añadir precalentado (a 37 ° C, crítico) Accutase e incubar a 37 ° C durante 5-10min con agitación periódica para asegurarse de esferas de células madre neuronales disocia en células individuales (por NCSC fijación cultivadas, las células se incuban con Accutase a 37 ° C durante 5 min para asegurarse de que células madre del folículo del pelo desprendido). </li><li> Lavar y centrifugar las células dos veces durante 5 min a 200 xg, a temperatura ambiente en medio DMEM/F12 para eliminar cualquier solución Accutase restante. </li><li> Vuelva a suspender las células con medio de cultivo NCSC. </li><li> Para el cultivo flotante, poner las células en placas de fijación ultra-bajas. Para el cultivo de apego, puesto que las células pretratadas en placas de cultivo de tejidos. </li></ol> 6. Los resultados representativos Medio NCSC cultivo es suficiente para mantener hNCSCs en un estado indiferenciado sin la necesidad de células alimentadoras. Los queratinocitos no proliferan y gradualmente murió en el medio. Ciertas células redondas pequeñas se proliferan y forman agregados pequeños en suspensión después de 3 a 5 días. Estos agregados flotantes aumentó lentamente itamaño n, generados en tres dimensiones como estructuras de esfera, que denominamos esferas de cabello (Figura 3A). Si cultivaron en placas recubiertas, las CTCN se adhieren a la superficie y no forma ningún esferas. Los CTCN conectados crecen más rápido que en suspensión. Las células madre que forman la esfera-o adjunto expresan marcadores CTCN. Cuando los folículos pilosos son enteros culturas, las esferas se forman en el área correspondiente a la región protuberancia (Figura 3B). <img alt="Figura 1" src="/files/ftp_upload/3194/3194fig1.jpg" /> Figura 1. Desplumados anágena (A) y telógena (B) folículos pilosos. <img alt="Figura 2" src="/files/ftp_upload/3194/3194fig2.jpg" /> Figura 2. Imágenes representativas de análisis FACS de CTCN panel izquierdo:. Células son gated usando anticuerpos anti-CD271 y anti alphe-4 integrina anticuerpos. Panel derecho: las células están cerradas utilizando anticuerpos anti-CD271 y anti-HNK1. <img alt="Figura 3" src="/files/ftp_upload/3194/3194fig3.jpg" /> Figura 3. Morfología de las esferas de pelo (A) Morfología de esferas flotantes de pelo, panel izquierdo:. Baja energía, panel de la derecha:. Alta potencia (B) Morfología de la esfera de pelo in situ en la región protuberancia, panel izquierdo: una esfera de pelo en bulto telógeno, panel de la derecha: una esfera pelo en la región bulto de un folículo de cabello anágeno.

<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. 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No tenemos nada que revelar.

Los métodos de aislamiento de células y cultivo descritos son reproducible y robusto. Hemos generado CTCN de docenas de individuos a través de un amplio rango de edades. Aunque es mejor para procesar el tejido justo después de la cosecha de tejidos, se encontró que los tejidos del cuero cabelludo puede ser almacenado de forma segura en los medios de comunicación sobre hielo para el transporte durante la noche con un impacto mínimo sobre la viabilidad celular. Es importante tratar el t...

<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

Los folículos pilosos experimentan un crecimiento durante toda la vida y el ciclo del cabello es un proceso bien controlado que implica proliferación de células madre y la quiescencia. Bulto del pelo es un nicho bien caracterizado para las células madre adultas 1. Este segmento de la vaina radicular externa contiene un número de tipos diferentes de células madre, incluyendo células madre epiteliales, células melanocitos 2 madre 3 y de la cresta neural como las células madre 4-7. Los folículos pilosos son una fuente rica y accesible para los diferentes tipos de células madre humanas. Nosotros y otros han aislado células madre de la cresta neural (CTCN) de folículos pilosos humanos fetales y adultos 4,5. Estas células madre humanas son la etiqueta de retención de células y son capaces de auto-renovación a través de la división celular asimétrica in vitro. Ellos expresan marcadores neuronales inmaduras de células de la cresta, pero no marcadores de diferenciación. Nuestro estudio demostró que la expresión de perfiles que comparten un patrón de expresión génica similar con murino piel inmadura CRE neuralcélulas ST. Exhiben multipotencia clonal que puede dar lugar a linajes de células miogénicas, melanocíticos, y después neuronal en clonal in vitro de cultivo celular solo. Las células diferenciadas no sólo adquirir linaje marcadores específicos, sino también demostrar las funciones adecuadas en condiciones ex vivo. Además, estos CTCN mostrar potencial de diferenciación hacia linajes mesenquimales. Células neuronales diferenciadas pueden persistir en cerebro de ratón y retener marcadores neuronales de diferenciación. Se ha demostrado que el pelo del folículo derivado CTCN puede ayudar a la regeneración del nervio, y mejorar la función motora en los ratones trasplantados con estas células madre después de una lesión transección de la médula espinal 8. Además, los nervios periféricos han sido reparados con injertos de células madre 9, y la implantación de las células precursoras derivadas de la piel adyacentes a aplastadas nervios ciáticos se ha traducido en la remielinización 10. Por lo tanto, los folículos pilosos / piel CTCN derivados ya han mostrado resultados prometedores para regenerative terapia en modelos preclínicos. Reprogramación de células somáticas a madre pluripotentes inducidas (iPS) de células ha demostrado un enorme potencial para la medicina regenerativa. Sin embargo, existen todavía muchos problemas con las células iPS, en particular el efecto a largo plazo de la integración oncogén / virus y tumorigenicidad potencial de las células madre pluripotentes no han sido adecuadamente tratadas. Todavía hay muchos obstáculos que superar antes de que las células iPS pueden utilizarse para la medicina regenerativa. Mientras que las células madre adultas se sabe que son seguros y que se han usado clínicamente durante muchos años, tales como trasplante de médula ósea. Muchos pacientes se han beneficiado ya del tratamiento. Autólogo de células madre adultas son todavía células preferidas para el trasplante. Por lo tanto, las células adultas y de fácil acceso expandible madre en la piel humana / folículos pilosos son una valiosa fuente para la medicina regenerativa.

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

En este artículo se presenta un robusto protocolo para el aislamiento y cultivo de células de la cresta neural madre de folículos pilosos humanos.

Aislamiento y cultivo de células troncales de la cresta neural de los folículos del cabello humano

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

isolation-culture-neural-crest-stem-cells-from-human-hair

Research

JoVE Journal

Neuroscience

16.9K vistas.  School of Medicine, University of Pennsylvania. En este artículo se presenta un robusto protocolo para el aislamiento y cultivo de células de la cresta neural madre de folículos pilosos humanos.

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.

La producción de extracto de embrión de pollo para el Cultivo de murino crestas de células neurales madre

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

Investigación

Neurociencias

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.

Generación de las células troncales nerviosas de desechados Humanos tejido cortical fetal

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

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Aislamiento y cultivo de células humanas las papilas fungiformes gusto

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

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.

Aislamiento y cultivo de células neuronales de ratas embrionarias: un protocolo rápido

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.

Aislamiento y cultivo de células de la cresta neural de embriones murinos del Tubo Neural

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 ratón, dos culturas: Aislamiento y cultivo de adultos Neural las células madre de las dos zonas neurogénicos de ratones individuales

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.

Derivación-Feeder libres de Células Progenitoras de la cresta neural Pluripotentes Células Madre

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.

Aislamiento y cultivo de células madre adultas neural de la Zona subcallosal Ratón

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.

Desarrollo de un protocolo de refinado para Trans-escleral transplante Subretinal de células epiteliales del pigmento retiniano humano a ojos de rata

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

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Sencilla generación de una cultura de alto rendimiento de las neuronas inducidas de fibroblastos de piel humana adulta

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...