Nós gostaríamos de agradecer Marc Wozniak para a assistência de vídeo. Gostaríamos também de agradecer Sean Morrison na UT Southwestern para o protocolo original para a cultura de células de ratos NC. Este trabalho foi apoiado Vanderbilt University Medical Center Programa de Apoio Acadêmico e por concessões do NIH (HD36720 e HD036720 11S109-) eo 11GRNT7690040 AHA para PAL, bolsas predoctoral da AHA (0615209B) e NIH (NS065604) para NAM, e ERP foi apoiada por um treinamento NIH concessão T32HD007502.

1. Placas de Preparação <ol><li> Use técnica estéril em todos os momentos. </li><li> Preparar fibronectina (FN) por diluição de 100 ul de estoque humano FN plasma para um volume final de 3,3 mL em PBS de Dulbecco (DPBS). Concentração final é de 30 ug / mL e esta pode ser armazenada a 4 ° C durante 1 semana. </li><li> Cobrir parte inferior de cada poço de uma cultura de tecidos estéril quatro placa bem com uma solução de FN e deixar assentar durante 15 minutos. Certifique-se de toda a superfície é coberta. Tornar a mídia durante este tempo (passos 2 e 3). </li><li> Remover FN solução e permitir que as placas para secar. Suavemente lavar os poços com 500 uL de DMEM, remove DMEM, e adicionar 500 uL de auto-renovação (SR) médio (ver abaixo). Incubar a 37 ° C numa incubadora humidificada contendo 5 por cento de CO2. Complete esta hora aproximadamente um antes de dissecção de modo que as placas será seca antes da utilização. </li></ol> 2. Preparando Médio SR <ol><li> Para a cultura de NC vagal e troncoa partir de embriões (aproximadamente dez uma ninhada, dependendo de fundo genético da linha de rato), preparar 25 mL de meio de SR. Combinar 12,5 mL de baixa glicose DMEM, 7,5 ml de meio Neurobasal, 25 uL de ácido retinóico (117 uM de concentração final), e 25 uL de 2-mercaptoetanol (50 mM de concentração final). Misture bem. </li><li> Adicionar 3,75 ml Extracto de embrião de pinto, 250 uL de N suplemento de sal 2, 500 suplemento B27 uL e 250 uL de penicilina-estreptomicina (1% de concentração final). Filtrar o meio através de um filtro 0,22 uM. </li><li> Adicionar 10 uL IGF1 estéril (20 ug / mL concentração final) e 20 uL de bFGF estéril (20 ug / mL concentração final). Misturar por inversão. Armazenar a 4 ° C. </li></ol> 3. Preparação meio de lavagem <ol><li> Durante cerca de 10 embriões preparar 50 mL de meio de lavagem. Combinar 50 mg de BSA com 35 mL de baixa glicose DMEM, 15 ml de meio Neurobasal, e 500 uL de penicilina-estreptomicina (1% de concentração final). Filtro estéril com um 0,22filtro uM. </li></ol> 4. Preparando colagenase / dispase <ol><li> Adicionar 50 ul de 100 mg / mL de colagenase / dispase a 5 DPBS mL. Misture bem. </li><li> Filtro de seringa com um filtro 0,2 um e adicionar 1,5 mL em cada um dos três poços de uma placa de 12 bem. Pipetar cerca de 1 mL em meio de lavagem dos poços restantes. Guarde placa inteira em gelo até estar pronto para digerir tecido dissecado. </li><li> Cortar pontas de uma ponteira p20 e p1000 filtro com uma lâmina estéril. Cortar logo abaixo da aresta chanfrada da ponta. O ponto de corte p1000 irá ser usada para transferir o embrião inteiro, enquanto a p20 irá ser usada para transferir pedaços de tecido isolado. </li></ol> 5. Isolando vagal e Tubo Neural tronco de embriões 9,5 Dpc <ol><li> Para gravidezes programadas, as barragens com um plug vaginal são considerados 0,5 dpc ao meio-dia pela manhã a ficha é observado. Sacrifício e remover o útero de 9,5 dpc. </li><li> Remover o decidua a partir de útero e suavemente remove o embrião da decídua. Uma vez que experimentou com este protocolo, isolar 3-4 9,5 embriões Dpc em um momento em DPBS estéreis. Use técnica estéril e instrumentos de dissecção esterilizados. Instrumentos podem ser esterilizados por autoclave a esterilização por calor, ou por incubação em etanol. </li><li> Para anterior vagal NC: usando agulhas de insulina, cortar o tubo neural no placode meados ótica. Corte novamente na borda posterior do somito 4 º. Aparar tecido ventral para o tubo neural para remover os arcos da faringe e do coração. </li><li> Para NC tronco: usando agulhas de insulina, remover a porção do tubo neural entre 16-22 sómitos (ou o somito última se os embriões são developmentally mais cedo do que a fase somito 22). </li><li> Mantenha o saco vitelino e qualquer tecido remanescente embrionário para genotipagem. </li></ol> 6. A remoção de não-neural ectoderma e mesoderma <ol><li> Colocar o tubo neural contendo segmentos em colagenase / dispase à temperatura ambiente durante 10 minutoss. Lavar imediatamente em meio de lavagem. </li><li> Retornar ao tecido DPBS estéreis. Usando agulhas de insulina estéreis, remover suavemente a ectoderme não a partir do tecido neural e separar os sómitos longe do tubo neural. As últimas partes restantes da mesoderme a partir de tecido somito pode ser removido por trituração suavemente, utilizando um cut-down p20 ponta. Tenha cuidado para não danificar o tubo neural. Observe atentamente este processo durante a trituração. </li><li> Colocar o tubo neural isolado por meio de um segundo e terceiro lavagem de 30 segundos de meio de lavagem. </li><li> Lavar uma vez em meio de SR, e colocar o tubo neural isolado para o centro de um poço FN-revestido que foi previamente preparado (Passo 1,3). Humidificar a câmara de hipóxia com um prato de água estéril. Colocar a cápsula para dentro da câmara hipóxia e lavar a câmara de gás misturado com a 3% de O 2 (utilizar um tanque contendo uma mistura de 1% de O 2, 6% de CO 2, 93% N 2). </li><li> Sempre manuseie a câmara com muito cuidado para garantir thaexplantes t estão livres e permanecem no centro dos poços. </li><li> Incubar a 37 ° C. Resumo dos passos 5-6 é mostrado na Figura 1. </li></ol> 7. A remoção do Tubo Neural <ol><li> Após 24 horas de incubação, remover o tubo neural suavemente provocando a borda do tubo neural longe das células migratórias usando uma agulha de insulina estéril. Remover e descartar o tubo neural a partir do meio usando um corte p20 estéril como no passo 6,2 e substituir o meio com meio fresco SR (Figura 2). Isto é mais facilmente feito usando um microscópio invertido. </li></ol> 8. Os resultados representativos Após 24 horas de incubação a 37 ° C em condições de hipóxia, as células de NC ter migrado para longe do tubo neural em uma população quase puro (Figura 3a). Às vezes, menos do que as culturas ideais não trará conseqüências robustos. Por exemplo, é possível que, após 24 horas tele tubo neural terá enrolado sobre si próprio e à NC não irá migrar para longe do tubo neural (Figura 3b). Ocasionalmente, o tubo neural não irá anexar as placas fibronectina revestidos. Em nossa experiência, sub-óptima migração NC ou problemas com a fixação do tubo neural podem ser adversamente afetados por condições de normóxia ou concentração da fibronectina, respectivamente. A actividade enzimática da colagenase / dispase varia ligeiramente por lote e tempo de digestão deve ser ajustada adequadamente, no entanto, não digerir o tecido mais tempo do que quinze minutos. Overdigestion do tubo neural contendo tecido em colagenase / dispase também irá resultar em conseqüências deficientes. Se o tecido somito não é facilmente removido do tubo neural após incubação em colagenase / dispase, o tubo neural pode ser incubada durante mais de dez minutos. Às vezes, o tubo neural não irá anexar ao substrato. Se este for o caso, verifique a fibronecconcentração de estanho e as condições de hipoxia. Embora as condições normóxicas pode ser utilizado para a cultura de tipo selvagem NC, condições hipóxicas imitar mais de perto o ambiente in vivo 29,30. Em nossa experiência, condições de hipóxia tornou-se crítica quando a cultura mutante NC. Por exemplo, quando FOXD3 mutante NC foram cultivadas em condições normóxicas, tronco NC tinha uma excrescência célula grandemente reduzida em comparação com os controlos. Esta disparidade em tamanho excrescência foi removido quando os explantes foram cultivados em condições de hipóxia (Figura 4). Além disso, quando do tipo selvagem do tubo neural explantes foram cultivados em normóxia, o número de células positivas para caspase foi maior do que aquela de explantes semelhantes cultivadas em hipoxia (dados não mostrados). Ao manter toda a cultura NC em hipóxia, as comparações podem mais facilmente ser feito entre a dinâmica de controle e culturas mutantes. <img src="/files/ftp_upload/4134/4134fig1.jpg" alt="A Figura 1" /> <strong&gt; Figura 1. Esquemática geral de isolamento NC. A) Dissecar regiões de interesse a partir do embrião. B) Digest tubo neural em colagenase / dispase por dez minutos (não ultrapassar quinze minutos). C) Lavar em meio de lavagem. D) Dissecar longe do ectoderma não-neural e mesoderma. EF) Lavar duas vezes em meio de lavagem. G Plate) em meio de auto-renovação. Incubar a 37 ° C em 3% de O2 condições hipóxicas. <img src="/files/ftp_upload/4134/4134fig2.jpg" alt="A Figura 2" /> Figura 2. Remoção gradual do tubo neural de explante. O tubo neural deve ser removida depois de 24-48 horas para evitar a contaminação com não-NC células. A) Observe o limite entre o tubo neural ea excrescência NC (linha sólida). B) O corte ao longo da borda do tubo neural com uma agulha de insulina. C) Descartar o tubo neural e substituí-meio com meio fresco auto-renovação. A linha a tracejado indica o grau de crescimento. Sigla: NT, o tubo neural. Figura 3. Exemplos de resultados representativos. A) excrescência explante típica após 24 horas de incubação em meio de auto-renovação em condições de hipóxia (linha a tracejado indica o grau de crescimento). B) Magnified vista da excrescência NC após 48 horas em cultura. C) a cultura Menos ideal com rendimento baixo crescimento. A e C foram cultivadas nas mesmas condições. Imagens demonstrar a gama natural em robustez cultura. Isto pode ser afectada pela eficiência do isolamento do tubo neural, a concentração de FN, condições de hipóxia, eo tempo em colagenase / dispase digestões. <img src="/files/ftp_upload/4134/4134fig4.jpg" alt="A Figura 4" /> Figura 4. Em análises in vitro de culturas de explante NF em normóxia contra a hipoxia. Controle (tipo selvagem), as células migraram a partir de explantes de NC tubo neural após 48 horas em condições de cultura normóxicas. Em contraste, FOXD3 mutanteNC tinha bastante reduzido conseqüências celulares em normóxia (bordas contornos marca vermelha das conseqüências NC). Quando explantes comparáveis ​​foram cultivadas sob condições de hipóxia, FOXD3 explantes mutantes NC cresceu comparavelmente aos controlos, permitindo que as análises subsequentes. Nota, este comportamento correlacionou bem com o comportamento de FOXD3 mutante NC in vivo.

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Não temos nada a divulgar.

Atenção especial deve ser dada à fase de desenvolvimento do embrião para garantir o sucesso desta abordagem. Contando somitos de embriões de rato é fundamental tanto para a fase correspondente embriões dentro de uma maca e determinar as regiões corretas do tubo neural para o isolamento. Uma variação de um ou dois sómitos entre os embriões está dentro de uma gama razoável de tempo de desenvolvimento, dependendo da resolução da experiência realizada. Um embrião entre 9 e 9,5 dpc terá entre 17 e 25 somitos. Se o embrião tem mais de 25 somitos, o NF é mais avançada no tempo de desenvolvimento, e excrescências NF será menos robusto em cultura. Estadiamento embriões com base no número somito facilita precisão em experimentos quando o estágio de desenvolvimento do embrião e, portanto, NC, pode influenciar o resultado dos experimentos. Em um embrião DPC 9,5, o NC vagal migra a partir do tubo neural em dorsal definidos anterior-posterior limites: a partir do placode meados de ótica para somito 7.Do mesmo modo, tronco NC migra a partir do tubo neural em níveis somito 8 a somito 24. Para isolar populações distintas de células progenitoras NC como descrito neste protocolo, discreto sub-regiões dentro de cada um desses domínios são isolados. A região do tubo neural usado irá variar com base na concepção da experiência e da região anterior-posterior do NC ser estudado. As condições de cultura acima descrito, incluindo o meio e as condições de incubação, são especificamente adaptado para a cultura de NC murino. SR média semelhante foi utilizada pela primeira vez para a cultura de rato NC 11. Nas nossas mãos, este meio de rato, com a adição de meio Neurobasal, produz uma excrescência robusto de progenitores de murídeo NC. Além disso, diferentemente de trabalho anteriores de cultura de células de aves NF, uma camada alimentadora é desnecessária para a cultura do NC mamíferos utilizando este método 11,13,21,22. Embora existam inúmeros protocolos que detalham a cultura NC tanto de aves e mamíferos em normóxica conditi<html> ons 9,22, que 23 (Figura 4), ​​juntamente com outras pessoas no campo 20, descobriram que a cultura de células murinas NF em condições de hipóxia, como descrito aqui sobrevivência muito aids e auto-renovação, presumivelmente devido a hipóxia mais imita os níveis de oxigênio fisiológicos dentro do embrião 20,29,30. A avaliação da expressão de marcadores moleculares facilita grandemente a identificação de células NC e seus derivados diferenciadas. Estes incluem expressão de FOXD3, p75, e SOX10 para as células estaminais NC. Marcadores de NC diferenciada incluem músculo liso alfa-actina (SMA) para miofibroblastos, proteína glial fibrilar ácida (GFAP) para glia, microftalmia associada fator de transcrição (MITF) para os melanócitos, e beta-tubulina III, produto protéico do gene 9,5 (PGP9.5 ), periferina e para os neurônios. Estes marcadores podem ser usados ​​para determinar a eficiência do explante e diferenciação de progenitores NC. conteúdo &quot;&gt; Uma vez que esta técnica é dominada, NC cultivadas pode ser usado em uma variedade de ensaios, incluindo a quantificação da proliferação, a morte celular, a dinâmica de migração, e / ou diferenciação de estado. NC pode também ser cultivadas clonalmente para investigar a auto-renovação ou multipotency de progenitores individuais NF 23. Depois de o tubo neural é removido das culturas, dissociar as células da CN em 100 ul de tripsina-EDTA (0,25%) durante exactamente 5 minutos a 37 ° C. Extingue Tripsina-EDTA em meio de lavagem em excesso (8-10 ml), adicionando 800 ul por poço e transferindo para tubos de 15 mL individuais contendo meio de lavagem. suavemente centrifugar as células a 150 xg durante 3 minutos, remover o sobrenadante e ressuspender o pellet em 1 célula médio SR mL. contagem de células utilizando um hemocitómetro e placa-los em uma densidade de 25 células / cm 2. Estas colônias pode ser passado seriadamente para ensaio de auto-renovação. Para multipotency ensaio, manter as células da CN em densidade clonal em meio SR para 6 dias e depois mudar para a diferenciaçãomédio (10 ng / mL de bFGF e extracto de embrião de 1% pinto). Cultura das células em meio de diferenciação em hipóxia por 8 dias antes da análise da composição colônia com marcadores moleculares como acima de 11. Em conclusão, este método para isolamento de células de NC a partir de embriões de ratos produz um alimentador de cultura livre de células aderentes premigratory NC sem o uso de FACS. Análise de experiências usando estas células podem ser facilmente quantificado. Embora essa técnica é simples, suas aplicações para o estudo das características NC de migração, auto-renovação e diferenciação são vastas. Além disso, isolando NC a partir de embriões de ratos geneticamente modificados permite o estudo directa de genes particulares e vias no contexto de NC a migração, a sobrevivência e / ou diferenciação.

<table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> DMEM (glicose baixa) </td><td> Gibco / Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> Médio Neurobasal </td><td> Gibco </td><td> 21103 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A3912-10G </td><td></td></tr><tr><td> DPBS </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> IGF1 </td><td> BD Biosciences </td><td> 354037 </td><td> Armazenar em 50 ug / ml alíquotas a -20 ° C. </td></tr><tr><td> bFGF </td><td> BD Biosciences </td><td> 354060 </td><td> Armazenar em 25 ug / mL alíquotas a -20 ° C. </td></tr><tr><td> A fibronectina </td><td> Gibco </td><td> 33016-015 </td><td> Armazenado em 1mg/mL aliquots a -20 ° C. </td></tr><tr><td> O ácido retinóico </td><td> Sigma </td><td> R2625 </td><td> Armazenar em 35 ug / ml alíquotas após reconstituição em etanol a -20 ° C. </td></tr><tr><td> 2-mercaptoetanol </td><td> Sigma </td><td> D-5637 </td><td></td></tr><tr><td> N suplemento 2 </td><td> Gibco </td><td> 17502-048 </td><td></td></tr><tr><td> B27 suplemento </td><td> Gibco </td><td> 17504-044 </td><td></td></tr><tr><td> Steriflip de filtros de 0,22 uM </td><td> Millipore </td><td> SCGP00525 </td><td></td></tr><tr><td> Penicilina-estreptomicina </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> 0,20 mM filtros </td><td> Corning </td><td> 431219 </td><td></td></tr><tr><td> Seringas (para a filtração) </td><td> BD Biosciences </td><td> 301604 </td><td></td></tr&gt; <tr><td> Quatro placas de poços </td><td> Thermo Fisher Scientific </td><td> 176740 </td><td></td></tr><tr><td> Colagenase / dispase </td><td> Roche </td><td> 269 ​​638 </td><td> Atividade varia por lote. Armazenar em 100 mg / ml alíquotas a -20 ° C. </td></tr><tr><td> Agulhas de insulina (29 manômetro ½) </td><td> Becton Dickson </td><td> 309306 </td><td></td></tr><tr><td> Câmara hipóxia </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Analisador de Oxigênio </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Fórceps # 5 </td><td> Belas Ferramentas Ciência </td><td></td><td> Para a remoção do útero e decídua. </td></tr><tr><td> Tripsina-EDTA (0,25%) </td><td> Gibco </td><td> 25200 </td><td></td></tr></tbody></table>

isolation and culture of neural crest cells from embryonic murine neural tube

O embrionário da crista neural (NC) é uma população de células progenitoras multipotentes que origina na face dorsal do tubo neural, sofre uma transição epitelial para mesenquimal (TEM) e migra ao longo do embrião, dando origem a diversos tipos celulares 1-3. NC também tem a capacidade única de influenciar a diferenciação e maturação de órgãos-alvo 4-6. Quando explantado in vitro, células progenitoras de NC submetidos a auto-renovação, migrar e diferenciam-se em uma variedade de tipos de tecidos incluindo neurónios, células gliais, células musculares lisas, cartilagem e do osso. Multipotency de NC foi descrita pela primeira vez a partir de explantes do tubo neural aviária 7-9. Em isolamento in vitro de células de NC facilita o estudo de NC dinâmica, incluindo a proliferação, migração e multipotency. Trabalho adicional nos sistemas de aves e de rato demonstraram que as células explantadas NC NC reter o seu potencial quando transplantadas de volta para o embrião 10-13. Como essas propriedades inerentes celulares são preservadas em progenitores explantadas NF, o ensaio de explante tubo neural fornece uma opção atrativa para o estudo da NC in vitro. Para alcançar uma melhor compreensão da NC mamíferos, muitos métodos têm sido utilizados para isolar populações NC. NC-derivados progenitores podem ser cultivadas a partir de pós-migratórias locais em ambos o embrião e adulto para estudar a dinâmica dos progenitores pós-migratórias NC 11,14-20, no entanto isolamento de células progenitoras de NC como eles emigram a partir do tubo neural fornece preservação óptima de NC célula potencial e propriedades migratórias 13,21,22. Alguns protocolos empregar separação de células activadas por fluorescência (FACS) para isolar uma população enriquecida para NC progenitores particulares 11,13,14,17. No entanto, quando se inicia com embriões na fase inicial, os números de células adequadas para as análises são difíceis de obter com FACS, complicando o isolamento de início NC populations a partir de embriões individuais. Aqui, descrevemos uma abordagem que não depende de FACS e resultados em uma população de aproximadamente 96% NC puro com base em uma Wnt1-Cre repórter linhagem ativado 23. O método aqui apresentado é uma adaptação de protocolos otimizados para a cultura do rato NC 11,13. As vantagens deste protocolo, em relação aos métodos anteriores são de que 1) as células não são cultivadas em uma camada alimentadora, 2) FACS não é necessário para obter uma população relativamente puro NC, 3) células premigratory NF são isolados e 4) os resultados são facilmente quantificado. Além disso, este protocolo pode ser utilizado para o isolamento de NC a partir de qualquer modelo de rato mutante, facilitando o estudo das características NF com diferentes manipulações genéticas. A limitação desta abordagem é que o NF é removido a partir do contexto do embrião, o que é conhecido para influenciar a sobrevivência, a migração ea diferenciação do NC 2,24-28.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

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

Isolamento de crista neural embrionário a partir do tubo neural facilita o uso de<em&gt; In vitro</em&gt; Métodos para estudar a migração, a auto-renovação, e multipotency da crista neural.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.1K visualizações.  Vanderbilt University Medical Center. Isolamento de crista neural embrionário a partir do tubo neural facilita o uso de In vitro Métodos para estudar a migração, a auto-renovação, e multipotency da crista neural.

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Neurociência

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 ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Estabelecer embrionárias de rato Cultura de Células-tronco neurais utilizando o ensaio Neurosphere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

Caracterização fisiológica, morfológica e neuroquímicos de Neurônios Modulada por Movimento

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Fechamento do tubo neural no embrião do rato Cultura Whole

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

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

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

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 ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reprodutível mouse esmagamento do nervo ciático e subsequente avaliação de Regeneração por Análise Whole Mount muscular

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Análise de migração da crista neural e Diferenciação por Cross-espécies de Transplantes

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

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 Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolamento e cultura de células endoteliais da Embryonic posencéfalo

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...