Este trabalho é apoiado pelo NIH concessão R01AR054593 e R01AR054593-S1 para Xu.

1. Preparação de placas de cultura de tecidos <ol><li> Revestimento bem cada uma com suficiente poli-D-lisina (PDL) para cobrir o fundo do poço. Permitir que as placas a secar ao capuz. </li><li> Depois de as cavidades são secos, enxaguar com água estéril, e aspirar. Permitir que as placas a secar ao capuz. </li><li> Quando pêlo, seca com fibronectina (que foi dissolvido em água BioWhittaker noite a 37 ° C a uma concentração de 1 mg em 6 ml). </li><li> Adicionar meio NCSC [DMEM/F12 95 ml, 1 ml Penn / Strep (P / S), 1 ml de N2, 2 ml B27, 100 ul de mercaptoetanol (2ME, 50 mM de estoque), bFGF (20 médio ng / ml), IGF -1 (20 ng / ml de meio) e EGF (20 ng / ml de meio)], antes de fibronectina seca nas placas, </li></ol> 2. Extrair células do folículo piloso de couro cabeludo humano <ol><li> Antes de iniciar o procedimento de isolamento NCSCs, uma necessidade de preparar os respectivos meios e reagentes (ver Tabela 1). </li><li> Pele do couro cabeludo fresco humano adulto de procedimentos facelift ou fetecido do couro cabeludo tal é recolhida, lavada com PBS contendo penicilina-estreptomicina. </li><li> A pele é então transferido para tubos de 50 ml e incubadas em DMEM com Dispase (10 mg / ml) durante a noite a 4 ° C. Incubação durante 2-4 horas a 37 ° C é também eficaz. Pedaços de pele deve ser uma largura máxima de 1 cm para permitir a penetração da enzima. </li><li> Transferir a pele para uma placa de Petri esterilizada, retirar cada fio a partir da pele, agarrando a haste do cabelo próximo da superfície da pele e puxando firmemente e suavemente. Os folículos do cabelo ou da morfologia mostram anágena (Figura 1A) ou telógena (Figura 1B). </li><li> Incubar os fragmentos isolados de folículos em 0,05% de tripsina-EDTA (Invitrogen) por 15-20 min à temperatura ambiente com agitação periódica, adicionar 4 ml de DMEM com FBS a 10% para parar a reacção. O epitélio folicular é tripsinizadas e filtrada através de filtro de 40 um para obter uma suspensão de célula única que contém as células de diferentes tamanhos e formas. </li><li> Girara 200 xg durante 5 min, descartar o sobrenadante cuidadosamente, re-suspender em 1 mL de PBS contendo soro a 2% (FBS). </li><li> Alternativamente, folículos pilosos depenados podem ser colocados em cultura sem digestão com tripsina a crescer esferas de cabelo in situ. </li></ol> 3. Isolamento de NCSCs folículo piloso Usando Fluxo de separação de células citométrico <ol><li> Folículo piloso suspensão de células individuais foram obtidos por digestão com tripsina e marcados com anticorpos contra CD271 (APC-conjugado) / HNK1 (conjugado com FITC) ou CD271 / integrina alfa 4 (PE-conjugado) durante 40 min em gelo, no escuro. </li><li> Centrifugar durante 5 minutos a 200 xg, à temperatura ambiente e aspirar o sobrenadante. </li><li> Ressuspender as células em PBS contendo soro a 2% (FBS), e antes da separação, a PI é adicionado a porta para fora as células mortas. </li><li> Executar separação de células por citometria de fluxo (FACS). Colete CD271 +, + HNK1 duplas células positivas ou CD271 +, + integrina alfa 4 duplas células positivas (Figura 2). </li><li> Depois de tipoing, CD271 +, + HNK1 duplas células positivas ou CD271 +, + integrina alfa 4 duplas células positivas são cultivadas em placas ultra-baixas de fixação no suporte NCSC </li></ol> 4. Cultura de NCSCs primários <ol><li> Cultura das células foliculares dissociados ou FACS ordenadas células em placas de ultra-baixas de fixação no meio NCSC, mudar de médio a cada dia. </li><li> Verifique cultura de células a cada dia sob o microscópio. NCSCs começará a formar pequenos agregados flutuantes após vários dias e bem formadas esferas em 2-5 semanas em cultura, dependendo da idade dos dadores (Figura 3A). Conseqüência aparecerá em poucos dias na região bojo e bem formados esferas in situ em várias semanas (Figura 3B) quando folículos inteiros são cultivadas no meio NCSC. </li></ol> 5. Expansão da NCSCs <ol><li> Lave as células uma vez com PBS. </li><li> Adicionar pré-aquecido (a 37 ° C, crítico) Accutase e incubar a 37 ° C durante 5-10min com agitação periodicamente para certificar-se de esferas de células-tronco neurais dissociada em única célula (para NCSC penhora de cultura, as células incubadora Accutase a 37 ° C por 5 minutos para ter certeza que as células-tronco do folículo de cabelo separado). </li><li> Lave as células duas vezes e centrifugar durante 5 minutos a 200 xg, à temperatura ambiente, em meio DMEM/F12 para remover qualquer solução restante Accutase. </li><li> Re-suspender as células com meio de cultura NCSC. </li><li> Para a cultura flutuante, colocar as células em placas de ultra-baixos de fixação. Para a cultura anexo, colocar as células em placas de cultura de tecidos pré-tratados. </li></ol> 6. Resultados representativos NCSC meio de cultura é suficiente para manter hNCSCs num estado indiferenciado, sem a necessidade de células de alimentação. Os queratinócitos não irão proliferar e gradualmente morreu no meio. Certas pequenas células redondas irão proliferar e formar pequenos agregados em suspensão após 3 a 5 dias. Estes agregados flutuantes aumentada lentamente in dimensões, gerados tridimensionais esfera estruturas semelhantes, que nós denominamos esferas de cabelo (Figura 3A). Se em cultura em placas revestidas, as NCSCs vai anexar à superfície e não formam quaisquer esferas. Os NCSCs anexados crescer mais rápido do que em suspensão. As células-tronco formadoras de esfera ou anexado expressam marcadores NCSCs. Quando folículos pilosos inteiras são as culturas, as esferas são formadas na área correspondente à região de protuberância (Figura 3B). <img alt="Figura 1" src="/files/ftp_upload/3194/3194fig1.jpg" /> Figura 1. Arrancado anágena (A) e telógena folículos (B) de cabelo. <img alt="Figura 2" src="/files/ftp_upload/3194/3194fig2.jpg" /> Figura 2. Imagens representativas de análise FACS de NCSCs painel esquerdo:. Células são gated usando anti-CD271 e anti-alphe quatro anticorpos integrina. Painel da direita: as células são gated utilizando anticorpos anti-CD271 e anti-HNK1. <img alt="Figura 3" src="/files/ftp_upload/3194/3194fig3.jpg" /> Figura 3. Morfologia das esferas de cabelo (A) Morfologia das esferas de cabelo flutuante, painel esquerdo:. Baixa potência, direito do painel:. Alta potência (B) Morfologia da esfera cabelo in situ na região da barriga, painel esquerdo: uma esfera de cabelo em bojo telógeno, painel direito: uma esfera de cabelo na região bojo de um folículo piloso anágena.

<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. 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Não temos nada a revelar.

Os métodos de isolamento e de cultura de células descritas são reprodutíveis e robusto. Geramos NCSCs de dezenas de indivíduos através de uma ampla faixa etária. Embora seja melhor para processar o tecido logo após a colheita de tecidos, descobrimos que os tecidos do couro cabeludo pode ser armazenado com segurança em meios de comunicação sobre o gelo para o transporte durante a noite com um impacto mínimo sobre a viabilidade celular. É importante para o tratamento do tecido do ...

<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

Folículos pilosos sofrem crescimento ao longo da vida e do ciclo do cabelo é um processo bem controlado envolvendo a proliferação de células-tronco e tranquilidade. Bojo de cabelo é um nicho bem caracterizada por células-tronco adultas 1. Este segmento da bainha externa da raiz contém um número de diferentes tipos de células estaminais, incluindo as células estaminais epiteliais, células 2 3 tronco dos melanócitos e da crista neural, como as células-tronco 4-7. Folículos pilosos representam uma fonte acessível e rico para diferentes tipos de células-tronco humanas. Nós e outros isolaram células-tronco da crista neural (NCSCs) de folículos capilares humanos fetais e adultos 4,5. Estas células estaminais humanas são etiquetas de retenção de células e são capazes de auto-renovação por divisão celular assimétrica in vitro. Expressam imaturos marcadores de células da crista neural mas não os marcadores de diferenciação. Nosso estudo mostrou perfil de expressão que eles compartilham um padrão de expressão de genes com semelhante murino pele imatura neural CREcélulas st. Eles exibem multipotência clonal que podem dar origem a linhagens de células miogênicas, melanocítica, e depois neuronal in vitro de cultura de células clonal única. As células diferenciadas não só adquirir linhagem marcadores específicos, mas também demonstram funções apropriadas em condições ex vivo. Além disso, estas mostram NCSCs potencial de diferenciação para linhagens mesenquimais. Diferenciadas de células neuronais pode persistir no cérebro do rato e reter marcadores de diferenciação neuronal. Tem sido demonstrado que folículo piloso derivado NCSCs pode ajudar recrescimento dos nervos, e que melhoram a função motora em murganhos transplantados com células estaminais após estas lesões da medula espinhal atravessa em 8. Além disso, os nervos periféricos foram reparadas com enxertos de células-tronco 9, e implantação de células precursoras derivadas da pele adjacentes aos nervos ciáticos esmagados resultou na remielinização 10. Portanto, o folículo piloso / pele NCSCs derivados já mostraram resultados promissores para rterapia egenerative em modelos pré-clínicos. Somática reprogramação celular para-tronco pluripotentes induzidas (iPS) células mostrou um enorme potencial para a medicina regenerativa. No entanto, ainda há questões com muitas células iPS, em particular o efeito de longo prazo do oncogene / vírus integração e tumorigenicidade potencial das células-tronco pluripotentes não foram adequadamente tratadas. Ainda há muitos obstáculos a serem superados antes que as células iPS podem ser utilizados para a medicina regenerativa. Considerando que as células estaminais adultas são conhecidos por serem seguros e de terem sido utilizados clinicamente durante muitos anos, como o transplante de medula óssea. Muitos pacientes já se beneficiaram com o tratamento. Autólogo de células-tronco adultas são ainda preferenciais células para transplante. Portanto, as células adultas de fácil acesso e expansível-tronco em humanos de pele / cabelo folículos são uma fonte valiosa para a 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

Este artigo apresenta um protocolo robusto para isolamento e cultura de células da crista neural-tronco de folículos capilares humanos.

Isolamento e Cultura de células da crista neural-tronco de folículos capilares humanos

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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Research

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Neuroscience

16.9K visualizações.  School of Medicine, University of Pennsylvania. Este artigo apresenta um protocolo robusto para isolamento e cultura de células da crista neural-tronco de folículos capilares humanos.

Vídeo: 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.

Produção de Extrato de embrião de galinha para o cultivo de células-tronco neurais murino Crest

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

Neurociência

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.

Geração de células-tronco neurais de Discarded tecido fetal humano Cortical

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.

Isolamento e Cultura de Células Papila fungiforme Humanos Taste

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.

Isolamento e cultivo de células neurais embrionárias de ratos: um 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.

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

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.

Um rato, duas culturas: isolamento e cultivo de células-tronco neurais Adulto de as duas zonas neurogênica Individual Ratos

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.

Derivação sem alimentador de células da crista neural progenitoras pluripotentes humanas de células-tronco

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 Cultura de células estaminais adultas neurais do Subcallosal Zona Rato

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.

Desenvolvimento de um protocolo refinado para Trans-escleral apresentam transplante de células epiteliais de pigmento Retinal humano nos olhos de rato

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.

Simples geração de uma cultura de alto rendimento de neurônios induzidas de fibroblastos de pele 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 ...