Agradecemos Denis Flipo para o conselho de processamento e análise de imagem, e David W. Silversides em cujo laboratório a linha de rato Gata4p [5kb]-GFP foi gerada. Pesquisas em laboratório Pilon é financiado pelo CIHR, NSERC, FRQS e FRQNT.

Declaração de Ética Experimentos envolvendo camundongos foram realizadas seguindo Conselho Canadense de diretrizes de cuidados com animais para o cuidado e manipulação de animais utilizados em pesquisas médicas. Protocolos que envolvem a manipulação de animais foram aprovados pelo comitê de ética institucional da Universidade do Quebec em Montreal (Comité Institutionnel de Proteção des Animaux; número de referência 0512-R3-650-0513). 1. Preparação de Colágeno Gel Trabalhar de forma estéril, sob uma capa de cultura de tecidos. <ol><li> Prepare completa 5x DMEM (meio essencial modificado de Eagle), incluindo antibióticos padrão. Dissolve-se 3,37 g de pó de DMEM e 0,925 g de NaHCO3 em 20 ml de água. Esterilizar por passagem através de um filtro de 0,22 um. Adicionar 2,5 ml de solução estéril de 100x de penicilina / estreptomicina e 25 ml de soro fetal bovino inactivado pelo calor estéril. Armazenar a 4 ° C. </li><li> No gelo, misture 800 mLO colagénio de solução I (3,77 mg / ml em 0,02 N de ácido acético, esterilizada por filtração), 600 ul de DMEM completo 5x e 17 ul de NaOH 1 N. Dilui-se com água estéril para um volume final de 3 ml. Incluir fatores de crescimento relevantes na mistura. Normalmente, utilizar o GDNF a 10 ng / ml para estimular a migração NCC entérico. </li><li> Depositar cerca de 480 mL em cada poço de uma única linha de uma placa de 24 poços. Evite bolhas. As restantes linhas pode ser usado para testar os efeitos de outros factores de crescimento sobre a migração das células. </li><li> Deixe o colagénio polimerizam, pelo menos, 1 hora numa incubadora esterilizada, a 37 ° C. </li></ol> 2. Dissecção de Animais 16 <ol><li> Configure acasalamentos e verificar se há plugs vaginais na manhã seguinte. E0.5 Day sendo meio-dia no dia a ficha vaginal for encontrado, isolar a fêmea e esperar 12 dias até E12.5. </li><li> Anestesiar ratos grávida com isoflurano e eutanásia por inalação de CO 2. </li><li> Pulverizar o mouse com etanol 70%. Levante as s abdômenparentes e abrir a cavidade abdominal com uma tesoura de dissecação. </li><li> Remover o útero para uma placa de Petri de vidro cheio com PBS gelado (soro fisiológico tamponado com fosfato). Corte o útero transversalmente entre inchaços deciduum individuais para isolar cada local de implantação do embrião. </li><li> Trabalhar em cada local de implantação separadamente em outra placa de Petri de vidro cheio de PBS gelado. Sob um microscópio de dissecação, utilizar uma pinça fina para remover a camada muscular do útero. </li><li> Abrir o saco vitelino visceral e âmnio para revelar o embrião. Tome cuidado ao cortar os vasos sanguíneos se juntar o embrião para a placenta / visceral saco vitelino, como eles estão interligados com os intestinos em desenvolvimento. </li><li> Cortar a cabeça do embrião no pescoço. </li><li> Insira um fórceps fechadas na cavidade abdominal de um embrião, logo acima do fígado de cor vermelho escuro e deixar a pinça aberta de si mesmo (deixar de aplicar pressão) para fazer uma abertura transversal na cavidade abdominal. Puxe as pinças abertas para baixo em direçãoa extremidade posterior do embrião para abrir completamente o abdómen. </li><li> Pegue o tecido conjuntivo por trás do fígado e puxar as tripas para fora do abdômen, tomando cuidado para não quebrar o intestino (cólon é ligado ao ânus). </li><li> Cortar o cólon para libertar as entranhas do resto do embrião. O corte pode ser feito em qualquer local ao longo do cólon. Reserve a parte da cauda para mais tarde. </li><li> Tease do tecido conjuntivo a partir do ceco, em seguida, o resto dos intestinos. Tenha cuidado para não ferir os intestinos ao fazê-lo. </li><li> Cortar o fígado e estômago (na extremidade rostral do intestino delgado), bem como os mesonefros e cristas genitais, se algum estiver presente. </li><li> Isolar o intestino delgado. Mais uma vez, tome cuidado para não cortar o intestino. A partir de agora, a orientação rosrrocaudal do tecido intestinal pode ser monitorado usando a curvatura acentuada presente na extremidade rostral. Deixar o intestino delgado em PBS à temperatura ambiente durante um tempo tão curto quanto possível, antes de incama (veja o passo 3.3). </li><li> Por fim, registrar o número de somitos cauda para cada embrião para determinar com precisão o estágio de desenvolvimento embrionário. </li></ol> 3. Seccionamento das embrionárias Intestines <ol><li> Antes de prosseguir com a dissecção de embrião, derreter 1,5 g de agarose em 100 ml de PBS (solução salina tamponada com fosfato) e manter a 50 ° C. </li><li> Despeje agarose fundida num molde de incorporação (por exemplo, um sistema fechado de 2 ml tubo de microcentrífuga que foi cortado longitudinalmente para excisar a cerca de 1/4 da parede do tubo). Deixe a agarose arrefecer para cerca de 42-45 ° C, deve ser apenas ligeiramente quente ao toque. </li><li> Incorporar o intestino embrionário pouco antes das solidifica agarose (esta pode ser avaliada com fórceps dicas e irá ocorrer em torno de 36-38 ° C). Segurando o intestino com uma pinça pela extremidade rostral dobrado, puxá-lo muito lentamente através da agarose ao longo do comprimento do molde. Isso ajuda a manter o intestino reto, enquanto os conjuntos de agarose. Solte o temitir assim que começa a resistir a ser movido. Mantenha o controle da orientação rostral-caudal do intestino. </li><li> Coloque o molde no frigorífico 2-3 minutos, para assegurar que a agarose completamente definido. </li><li> Retire a agarose a partir do molde (deslizando-o para fora do tubo Eppendorf abertas). Com uma lâmina, retirar o excesso de agarose em ambas as extremidades, fazendo cortes perpendiculares para o intestino. </li><li> Cole a extremidade rostral do intestino / agarose a bloquear-se na fase de metal de um micrótomo de lâmina vibrante. Aparar o excesso de agarose sobre os lados do bloco de intestino / agarose. </li><li> Monte a fase de metal na câmara de micrótomo vibrando. Se necessário, ajustar o ângulo da fase de modo que o intestino é mais vertical possível (portanto, perpendicular à lâmina). Cobrir as amostras com PBS gelado. Traga a lâmina de micrótomo até alguns milímetros abaixo da superfície do buffer. </li><li> Fazer 200 mM vibratome transversais cortes do intestino caudal-mais pequenos, garantindo que each fatia agarose contém uma seção intestinal completo. </li></ol> 4. Cultura da Intestinal explantes <ol><li> Gentilmente depositar o fatias intestino / agarose recém-cortadas plana para os géis de colágeno, com uma pinça, colocando uma fatia para o meio de cada poço. </li><li> Incubar 3 dias a 37 ° C, em húmido 5% de CO2, para permitir a migração de NCC fora do explante. </li><li> Leve as fatias de intestino / agarose fora do gel de colágeno muito delicadamente com uma pinça. Tome cuidado para não danificar o gel abaixo. </li><li> Evitar tocar directamente o gel de colagénio, durante o tempo de incubação e de lavagem de passos subsequentes, a fim de garantir que o padrão de migração de células não é perturbado. Fixar com 500 mL de 4% PFA (paraformaldeído) (em PBS) por poço 1 hora à temperatura ambiente. </li><li> Substituir o fixador com 500 mL de DAPI (4 &#39;,6-diamidino-2-fenilindole) de solução (5 ug / ml em PBS) por poço e incubar 10 min a temperatura ambiente. </li><li> Lave cada poço 3xcom 500 ul de PBS durante 5 min. </li><li> Fotografar as células fluorescentes (canais GFP e DAPI) incorporados no gel de colágeno dentro de cada poço. </li></ol> 5. Análise de Imagem Fizemos uso extensivo de ImageJ 17 para processar e quantificar as imagens geradas após cultura explante. <ol><li> Comece imaginando um slide micrômetro com a mesma ampliação como as fotografias de células fluorescentes. Meça o comprimento de um mícron de pixels (utilizando a ferramenta linha reta). </li><li> Defina a escala (Analyze / Set Scale, o número de entrada de pixels / micron). </li><li> Para cada fotografia GFP fluorescência, mude o formato de imagem para 8 bits em tons de cinza (Image/Type/8-bit). </li><li> Ajuste a intensidade do sinal (Imagem / Ajustar / Brilho / Contraste). </li><li> Subtrair o ruído de fundo, se necessário (Processo / Subtrair fundo, ajustar o raio de bola rolando). </li><li> Definir um limite para destacar as células e aglomerados de células (Imagem / Ajustar / Threshold), on aplicar um divisor de águas para dividir as touceiras (Processo / Binário / Bacia Hidrográfica). </li><li> Analisar partículas para especificar regiões de interesse (ROI) e gerar estatísticas de número de células (Analisar / Analisar Partículas; tamanho conjunto mínimo de excluir restantes pixels). </li><li> Definir opções de medida para incluir o diâmetro de Feret (Analyze / Definir Medidas). </li><li> Grupo ROIs e medir como um todo para determinar o diâmetro de Feret, uma indicação de difusão celular (Análise / Ferramentas / ROI Gerente / Mais / OU, então Gerente de ROI / Adicionar e ROI Gerente / Measure). </li></ol>

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Os autores não têm nada a revelar.

Nós mostramos como nosso ex vivo explante técnica de cultura pode ser usada para quantificar com precisão entérico potencial de migração NCC na presença de GDNF. Essa quantificação precisa é muito facilitada usando 200 seções vibratome intestino m de espessura em vez de grandes pedaços de tamanho aproximado, como descrito anteriormente 11,12,15. Na verdade, isso nos permite trabalhar com um número razoável de células em um ambiente altamente reprodutível. De nota, a distribuição un...

Células da crista neural (NCC) são um tipo de célula transitória única de vertebrados que forma muitos derivados durante o desenvolvimento do embrião. Esta população de células surge na fronteira da placa neural, ao lado ectoderma não-neural 1. Durante neurulação, curvatura da placa neural NCC lugares ao longo do bordo dorsal do tubo neural formando. NCC então sofrer uma transição epitelial-mesenquimal, segregar e migrar para longe do tubo neural. NCC colonizar várias estruturas embrionárias, incluindo o tracto digestivo onde formam todo o sistema nervoso entérico (ENS), uma rede interligada de gânglios neurais embutido na parede do intestino. Como recentemente revisou de 2,3, muitos genes foram envolvidos no desenvolvimento desta estrutura intrincada. A maior parte do ENS é derivado de uma pequena piscina de NCC proveniente do tubo neural vagal (ou seja, em torno do potencial limite rombencéfalo / medula espinal) 4.Estes progenitores neurais chegar ao intestino anterior em torno do dia embrionário (e) 9,0 em ratos e depois migrar caudalmente dentro do mesênquima intestino até aproximadamente e15.0 para colonizar todo o intestino embrionárias. Um subconjunto menor de células progenitoras neurais cólon também é fornecida por sacral NCC, que invadem o intestino posterior na direcção oposta até ao ceco 4. Ambos vagal e sacral NCC requerem múltiplos migração, proliferação, sobrevivência e-sinais de promoção de diferenciação para garantir a formação completa do ENS. A este respeito, os modelos animais - especialmente ratos geneticamente modificados - têm sido fundamentais para a identificação de vários ligandos extracelulares essenciais: GDNF (factor neurotrófico derivado de células gliais),,, (proteínas morfogenéticas do osso) BMPs neurotrofina-3 endotelina-3, Netrin , assim como Sonic e indiano Hedgehog (Shh e Ihh) 5-10. Destes, o GDNF a sinalização através de RET receptor transmembranar da tirosina quinase (Rearranged durante a transfecção), é reconhecido como the caminho mais crítico para a atração e orientação do NCC e para dentro do intestino embrionário. O GDNF é secretada pelo mesênquima intestino e forma um gradiente rosrrocaudal espaço-temporalmente controlado, que é directamente quimioatratante para entérico NCC, que expressam RET 11,12. Entre outras funções, o ENS regula o movimento no interior do tracto digestivo através da sua interacção com o músculo liso na parede intestinal. Ausência de gânglios neural na região terminal dos resultados intestinais na doença de Hirschsprung: a contração tônica do segmento afetado leva ao bloqueio, a acumulação a montante do material digerido e distensão maciça do intestino e abdômen. Doença de Hirschsprung ocorre cerca de um em 5.000 nascidos vivos. O padrão de migração rostro-caudal de entérico NCC é acreditado para ser o principal fator de contribuição à etiologia da doença de Hirschsprung. O cólon, mais afastada da fonte de migrar NCC e última porção de bowel a ser colonizada, é mais suscetível a defeitos na formação ENS. De acordo com o seu papel crucial na migração NCC entérico, a interrupção da sinalização GDNF / RET é a principal causa genética conhecida da doença de Hirschsprung 13. Para melhor estudar NCC e desenvolvimento ENS, geramos uma linha de camundongo transgênico - chamado Gata4p [5kb]-GFP 14 - em que migratório NCC são rotulados com Proteína Fluorescente Verde (GFP). A seguir, aperfeiçoou um ensaio ex vivo, a migração celular, adaptado a partir de trabalhos publicados por outros grupos 11,12,15, que agora permite a quantificação precisa do potencial de migração entérico NCC na presença de vários factores de crescimento, tais como o GDNF.

<table><tbody><tr><td>DMEM powder</td><td>Wisent</td><td>219-010-XK</td><td>&amp;nbsp;</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Bioshop</td><td>SOB999</td><td>Biotechnology grade</td></tr><tr><td>Steriflip vacuum filtration system (0.22 micron)</td><td>EMD Millipore</td><td>SCGP00525</td><td>&amp;nbsp;</td></tr><tr><td>Penicilin-Streptomycin solution, 100x</td><td>Wisent</td><td>450-201-EL</td><td>&amp;nbsp;</td></tr><tr><td>Fetal bovine serum</td><td>Wisent</td><td>095-150</td><td>High quality grade</td></tr><tr><td>Collagen I</td><td>BD biosciences</td><td>354236</td><td>&amp;nbsp;</td></tr><tr><td>NaOH</td><td>Bioshop</td><td>SHY700</td><td>Diluted from 10 N stock then sterile-filtered</td></tr><tr><td>GDNF</td><td>Cedarlane</td><td>CLCYT305</td><td>&amp;nbsp;</td></tr><tr><td>Falcon 24-well Plate</td><td>BD biosciences</td><td>353047</td><td>&amp;nbsp;</td></tr><tr><td>Dissecting scissors</td><td>Fisher Scientific</td><td>089515</td><td>&amp;nbsp;</td></tr><tr><td>Glass Petri dish</td><td>VWR</td><td>89000-306</td><td>&amp;nbsp;</td></tr><tr><td>PBS</td><td>Sigma</td><td>P5493</td><td>Cell culture grade</td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>M125</td><td>&amp;nbsp;</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-20</td><td>&amp;nbsp;</td></tr><tr><td>Agarose</td><td>Bioshop</td><td>AGA001</td><td>Biotechnology grade</td></tr><tr><td>Surgical blade</td><td>Feather</td><td>21</td><td>&amp;nbsp;</td></tr><tr><td>All Purpose Instant Krazy Glue Pen</td><td>Krazy Glue</td><td>KG824</td><td>&amp;nbsp;</td></tr><tr><td>HM 650V Vibrating-Blade Microtome</td><td>Thermo Scientific</td><td>920110</td><td>&amp;nbsp;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>&amp;nbsp;</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9564</td><td>&amp;nbsp;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Table of specific reagents and equipment.&lt;/p&gt;</td></tr></tbody></table>

a quantitative cell migration assay for murine enteric neural progenitors

Células da crista neural (NCC) é uma população de células multipotentes transitória e que se origina a partir do tubo neural dorsal e migra extensivamente em todo o embrião de vertebrados em desenvolvimento. Além de proporcionar a glia e os neurónios periféricos, NCC gerar melanócitos, bem como a maior parte do esqueleto crânio-faciais. A migração e diferenciação NCC é controlada por uma combinação da sua origem axial ao longo do tubo neural e sua exposição a estímulos extracelulares regionalmente distintas. Tal contribuição de ligandos extracelulares é especialmente evidente durante a formação do sistema nervoso entérico (ENS), uma rede de complexo interligado de gânglios neurais que controla localmente (entre outras coisas) o movimento do músculo do intestino e motilidade intestinal. A maior parte do ENS é derivado a partir de um pequeno conjunto inicial de NCC que empreender um longo percurso, a fim de colonizar - num rostral para caudal moda - todo o comprimento do intestino prospectivo. Entre as várias vias de sinalização conhecidosinfluenciar colonização NCC entérico, GDNF / sinalização RET é reconhecido como o mais importante. Na verdade, a secreção de espaço-temporalmente controlada do RET ligante GDNF pelo mesênquima intestino é o principal responsável para a atração e orientação de expressar-RET entérico NCC para e dentro do intestino embrionário. Aqui, nós descrevemos um ensaio ex vivo, a migração de células, fazendo uso de uma linha de ratinho transgénico possuindo marcado por fluorescência NCC, que permite a quantificação precisa do potencial de migração entérico NCC na presença de vários factores de crescimento, incluindo o GDNF.

Os seguintes resultados são representativos do que pode ser obtida com a técnica descrita aqui (Figura 1). O uso de factores de crescimento (isto é, de GDNF) estimula a migração dos entérico NCC fora do explante intestinal e no gel de colagénio-expressando GFP (Figura 2). Embora algumas células sair do explante, na ausência de factores de crescimento, estes não são mais marcadas com GFP e representam entrada passiva. É necessário remover a fatia intestinal a partir...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Apresenta-se um ensaio de migração de células ex vivo que permite a quantificação precisa do potencial de migração de células de crista neural entérico na presença de vários factores de crescimento.

Um ensaio de migração celular quantitativo para murinos progenitores neurais entéricas

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

9.2K visualizações.  UQAM.  Apresenta-se um ensaio de migração de células ex vivo que permite a quantificação precisa do potencial de migração de células de crista neural entérico na presença de vários factores de crescimento. 

Vídeo: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Campo elétrico controlado de migração dirigida de células progenitoras neurais em ambientes 2D e 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Bioengenharia

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Um Modelo de enzimas e Cultura de Células-Tronco Neural Serum-livre para EMT Investigation Adequado para Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Biologia do Desenvolvimento

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Avaliar motilidade celular Neural Stem Usando um dispositivo Agarose Gel baseado microfluídicos

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Biologia

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 ...

Neurociência

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

Um Ensaio Galvanotaxis para Análise de Precursores Neurais Cinética migração celular em um campo aplicado externamente Corrente Contínua Elétrica

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Detecção rápida de desenvolvimento neurológico fenótipos nas células precursoras neurais humanas (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Injeção sistêmica de células-tronco neurais / progenitoras em ratos com EAE crónica

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Imunologia e Infecção

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

Nucleofection de roedores Neuroblastos para estudar neuroblasto Migração<em&gt; In vitro</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Migração, Quimio-Atração e Ensaios de Cocultura para Células Endoteliais derivadas de células-tronco humanas e neurônios gabaergicos

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Visualizando a dinâmica das células neuronais e gliais no cérebro de camundongos em desenvolvimento

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Autor em destaque: Explorando a migração celular e os papéis dos genes no cérebro em desenvolvimento

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

Imagem de lapso de tempo de neurônios migratórios e progenitores gliais em fatias de cérebro de camundongo embrionário

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...