Name: isolation and expansion of adult canine hippocampal neural precursors
Uploaded: 2016-11-29T15:00:00.000+00:00
Duration: 9 min 37 s
Description: Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia grants (#568969 and 1004152).

De acordo com o New South Wales, Austrália lei, o tecido cerebral post mortem foi adquirida a partir cães adultos sacrificados por razões não relacionadas com o estudo. 1. Preparação do Meio de Cultura <ol><li> Prepara-se uma solução de gelatina a 0,1% por adição de 0,1 g de gelatina em pó a 100 ml de água destilada e agitando a 37 ° C até se dissolver. Esterilizar a solução por irradiação UV durante 15 min. Este meio pode então ser armazenado a 4 ° C durante até 1 mês. </li><li> Prepara-se uma proporção de 3: 1 de Dulbecco Modified Eagle Médium (DMEM) e mistura de nutriente F12 pela adição de 167 mL de F-12 a 500 ml de DMEM (4,5 g / L D-glucose). Adicionar 6,7 ml de solução de Penicilina / Estreptomicina (para uma concentração final de 1%). Este suporte de dados pode ser armazenada no escuro a 4 ° C durante até 1 mês. </li><li> Preparar 500 ml de meio de soro através da combinação de 440 mL de meio DMEM (4,5 g / L D-glucose), 5 mL de L-alanil-L-glutamina dipeptídeo (200 mM), 5 mL de Penicilina / Estreptomicina e 50 ml de bovin fetale soro (FBS). Este suporte de dados pode ser armazenada no escuro a 4 ° C durante até 1 mês. </li><li> meio de crescimento completo consiste em Neural Stem Cell (NSC) Meio Basal e proliferação Neural Suplemento-A (NS-A), 2% de FBS, 2 ug / ml de heparina, 20 ng / de factor de crescimento epidérmico ml (EGF) e 10 ng / mL factor de crescimento de fibroblastos básico (bFGF). Adicione este meio fresco, imediatamente antes da dissecação do cérebro, e aquecer a 37 ° C num banho de água. </li></ol> 2. Extração de cérebro <ol><li> Comece a extração dentro de 6 horas post mortem, esterilizando a superfície dorsal da cabeça do cão usando iodopovidona. </li><li> Utilizando um bisturi, fazer uma incisão longitudinal ao longo da linha média sagital, por toda a superfície dorsal do crânio, para expor os músculos subjacentes masséter. O comprimento deste corte irá depender da espécie e idade do cão. </li><li> Nas fronteiras rostral e caudal do crânio, faça adicional de 5 - 10 cm cortes perpendiculares em ambos os lados, passeing atrás dos olhos e ouvidos, respectivamente, para permitir que a pele a ser totalmente refletida. </li><li> Usando um bisturi, separar os músculos masseter de seu apego à crista sagital do crânio e raspar qualquer tecido conjuntivo remanescente do crânio. </li><li> Usando um osso oscilante viu cortar a tampa do crânio em uma linha circunferencial. Usando uma sonda ou bisturi cortar quaisquer anexos remanescentes com a dura-máter, e, em seguida, retire cuidadosamente a tampa do crânio para expor a superfície dorsal do cérebro. Cuidado: A espessura do osso varia em diferentes regiões do crânio, assim que o cuidado deve ser exercido para não penetrar muito profundamente e danificar o cérebro subjacente. </li><li> Separar a medula espinal por inserção de um bisturi entre as vértebras cervicais superiores. </li><li> Com uma mão para levantar suavemente o cérebro, usar um bisturi para libertar cuidadosamente o cérebro do crânio e fossas cortar os nervos e os vasos sanguíneos de ligação situadas no seu lado ventral. Levante cuidadosamente a bchuva para fora do crânio e para um recipiente de DPBS. Cuidado: Os grandes bulbos olfativos do cérebro canino pode estender-se profundamente na fossa craniana anterior. Deve ser tomado cuidado ao remover o cérebro de modo a preservar estas estruturas. </li></ol> 3. hipocampo Dissection <ol><li> Lavar o cérebro em solução salina tamponada com fosfato de Dulbecco (DPBS) para remover todo o sangue e colocá-lo lado dorsal para baixo sobre uma placa de Petri de 150 mM. </li><li> Lentamente bissectar o cérebro longitudinalmente através do plano médio-sagital utilizando uma faca cérebro molhado e uma única fatia que utiliza o comprimento total da lâmina. Não aplique pressão adicional, ou usar um movimento de corte, pois isso pode danificar o tecido. </li><li> Colocando cada medial hemisfério superfície para cima, dissecar o hipocampo utilizando um bisturi e pinça fina, e transferi-lo para um prato de cultura de tecidos de 35 mm. </li></ol> 4. Isolamento de células neurais precursoras <ol><li> Picar o tamostras de problema usando uma lâmina de bisturi em aproximadamente 1 mm 3 peças. </li><li> Transferir o tecido em um tubo de 15 ml, adicionar 2 ml de tripsina a 0,1% de EDTA, e incuba-se durante 7 minutos num banho de água a 37 ° C. </li><li> Pare a reacção enzimática pela adição de 4 ml de meio de soro para o tubo. Pellet da suspensão por centrifugação a 100 xg durante 7 minutos e remover o sobrenadante. </li><li> Ressuspenso o sedimento em 300 mL de DPBS, e mecanicamente dissociados cuidado pipetando para cima e para baixo, usando uma ponteira 1.000 l, até se formar um homogeneizado lisas. </li><li> Adicionar 14 ml de meio de soro ao tubo passar e a suspensão de células através de um coador de células de 40 ^ m. Pellet da suspensão por centrifugação a 100 xg durante 7 minutos, remover o sobrenadante e ressuspender em meio de crescimento completo. </li></ol> 5. Cultura Neurosphere <ol><li> A partir da suspensão de células obtida no isolamento, contar o número de células viáveis ​​utilizando a exclusão de tripano de corante azul e uma hemocitômetro. </li><li> Dilui-se a suspensão de células em meio de crescimento completo e sementes a 1 x 10 5 células / cm2 em poços não revestidos de uma placa de 6 poços. </li><li> Incubar a 37 ° C, 5% de CO 2 e&gt; 95% de humidade durante 14 - 28 dias, substituir 50% do meio de crescimento a cada 7 dias. Medir o diâmetro das neuroesferas regularmente. Se for permitido que crescem para além de 100 um de diâmetro, a morte celular e diferenciação espontânea pode ocorrer dentro das neuroesferas. </li></ol> 6. Neurosphere Passage <ol><li> Para passagem como uma cultura flutuante, quando as neuroesferas atingir cerca de 100 um de diâmetro, combinar todos os meios neuroesfera contendo em um único tubo. Sedimentar por centrifugação a 100 xg durante 7 minutos e remover o sobrenadante. </li><li> Ressuspender o sedimento em 1 ml de 0,1% de tripsina EDTA e incubar durante 7 minutos num banho de água a 37 ° C. Pipeta suavemente para cima e para baixo de 100 vezes, utilizando uma ponta de pipeta de 200 mL, para garantir dissociatisobre as neuroesferas. </li><li> Pare a reacção enzimática por adição de 5 ml de meio de soro para o tubo e centrifugar a 100 xg durante 7 minutos. </li><li> Remover o sobrenadante, ressuspender o sedimento celular em 1 ml de meio de crescimento completo e contar o número de células viáveis ​​utilizando azul de tripano de corante de exclusão e um hemocitómetro. </li><li> Dilui-se a suspensão de células em meio de crescimento completo e sementes a 1 x 10 5 células / cm2 em poços não revestidos de uma placa de 6 poços. Incubar a 37 ° C, 5% de CO 2 e&gt; 95% de humidade durante 14 - 28 dias, a substituição de 50% dos meios de crescimento a cada 7 dias. </li></ol> 7. Aderente Cultura de células neuronais precursoras <ol><li> Adicionar um volume suficiente de solução de gelatina a 0,1% para cobrir a superfície de um prato de cultura de tecidos e cura em estufa a 37 ° C durante 1 h antes de processar as neuroesferas para cultura aderente. </li><li> A partir da cultura neurosphere flutuante, combinar todas as mídias em um único tubo. Agregar as neurospheres por centrifugação a 100 xg durante 7 minutos e remover o sobrenadante. </li><li> Ressuspender o sedimento em 1 ml de 0,1% de tripsina EDTA e incubar durante 7 minutos num banho de água a 37 ° C. Pipeta suavemente para cima e para baixo de 100 vezes, utilizando uma ponta de pipeta de 200 mL. </li><li> Adicionar 5 ml de meio de soro para interromper a reacção enzimática e centrifugar a 100 xg durante 7 minutos. Descartar o sobrenadante e ressuspender o sedimento de células em 1 ml de meio de crescimento completo e contar o número de células viáveis ​​utilizando azul de tripano de corante de exclusão e um hemocitómetro. </li><li> Dilui-se a suspensão de células em meio de crescimento completo, remover a gelatina a partir dos frascos de cultura de tecidos e semear as células a 1 x 10 4 células / cm2. </li><li> Incubar as células a 37 ° C, 5% de CO 2 e&gt; 95% de humidade. Substituir a mídia com, aquecido, meios de crescimento frescos completas a cada três dias até que a cultura atinge aproximadamente 80% de confluência (após aproximadamente 7 dias). </li></ol> 8. Neural Colony Forming Assay <ol><li> Combine celular componentes de mídia (NCFC) ensaio de formação Neural Colony: meio de soro NCFC livre, Proliferação NS-A, e solução de colágeno (3 mg / ml). Suplemento este meio com 2 ug / ml de heparina, 20 ng / ml de EGF, e 10 ng / ml de bFGF. </li><li> Após dissociação de tecido do hipocampo ressuspender as células em colónia neural formando meio de ensaio, pré-aquecido a 37 ° C num banho de água. Semear as células a uma densidade clonal de 1,1 x 10 5 culas / ml num prato de cultura de 35 mm. </li><li> Incubar as células nesta matriz de colagénio semi-sólido a 37 ° C, 5% de CO 2 e&gt; 95% de humidade. Cultura de 28 dias, a substituição de 50% do meio de crescimento a cada 7 dias. </li></ol> 9. A diferenciação de células neuronais precursoras <ol><li> Adicionar um volume suficiente de 5 ug / cm solução laminina 2 para cobrir a superfície de um prato de cultura de tecidos e cura em estufa a 37 ° C durante 24 h antes do processamento das células de diferenciaçãoção. Nota: Remover a solução laminina e enxaguar a superfície revestida por duas vezes em DPBS imediatamente antes da semeadura. </li><li> Quando passaging, a semente da suspensão de células a 1 x 10 4 células / cm2 em DMEM-F12 (3: 1) meios (pré-aquecido a 37 ° C) suplementado com 20 ng / ml de EGF e 40 ng / ml de bFGF para o laminina revestido prato de cultura. </li><li> Incubar as células a 37 ° C, 5% de CO 2 e&gt; 95% de humidade e permitir que as células a proliferar durante 3 dias. </li><li> Para diferenciar as células precursoras neurais aderentes, depois de 3 dias substituir os meios de comunicação com meios de diferenciação contendo DMEM-F12 (3: 1) meios de pré-aquecido a 37 ° C, suplementada com 10 ng / ml cérebro factor neurotrófico derivado (BDNF). </li><li> Durante a diferenciação substituir 50% do meio a cada 3 dias. A diferenciação ocorre gradualmente ao longo de cerca de 21-28 dias. Cuidado: Uma vez que as células tenham começado a diferenciar-se, substituir apenas 50% dos meios de comunicação em uma TI-me como a exposição ao ar podem ter um efeito prejudicial na saúde das células. </li></ol>

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A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+enhances+neuronal+proliferation+and+astroglial+differentiation+in+cultures+of+CNS+stem+cell-derived+precursors.">Retinoic acid enhances neuronal proliferation and astroglial differentiation in cultures of CNS stem cell-derived precursors.</a> J Neurobiol. 37 (2), 281-290 (1998).</li><li>Araujo, D. M., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+effects+of+interleukin-4,-7+and-8+on+hippocampal+neuronal+cultures:+potential+involvement+of+glial-derived+factors.">Trophic effects of interleukin-4,-7 and-8 on hippocampal neuronal cultures: potential involvement of glial-derived factors.</a> Brain Res. 600 (1), 49-55 (1993).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Singec, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nat Methods. 3 (10), 801-806 (2006).</li><li>Conti, L., 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=Niche-independent+symmetrical+self-renewal+of+a+mammalian+tissue+stem+cell.">Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.</a> PLoS Biol. 3 (9), 283 (2005).</li><li>Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+neural+stem+(NS)+cells+from+fetal+and+adult+forebrain.">Adherent neural stem (NS) cells from fetal and adult forebrain.</a> Cereb Cortex. 16, 112-120 (2006).</li><li>Ma, W., 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=Cell-extracellular+matrix+interactions+regulate+neural+differentiation+of+human+embryonic+stem+cells.">Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.</a> BMC Dev Biol. 8 (90), 1-13 (2008).</li><li>Goffredo, D., 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=Setting+the+conditions+for+efficient,+robust+and+reproducible+generation+of+functionally+active+neurons+from+adult+subventricular+zone-derived+neural+stem+cells.">Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells.</a> Cell Death Differ. 15 (12), 1847-1856 (2008).</li></ol>

The authors have nothing to disclose.

Os protocolos aqui descritos são optimizados para manter condições favoráveis ​​de cultura para maximizar a viabilidade celular. A velocidade e cuidado durante a extração, isolamento e expansão é de importância crítica. Um passo fundamental para o estabelecimento de expansão monocamada aderente é a dissociação eficaz das neuroesferas primárias. Passagem seguinte, insuficientemente dissociada neurospheres pode gerar neurospheres flutuantes secundárias. Durante a mudança de mídia, estes neurospheres ...

Regional variations in the rate of neurogenesis have been observed along the dorsoventral axis of the rodent hippocampus1,2. Furthermore, the rates of hippocampal neurogenesis also show distinct inter-species variation, with precursor cell turnover in the subgranular zone shown to be significantly lower in adult humans than in rodents3-5. Inter-species differences in hippocampal structural anatomy may be relevant here, as it has been postulated that neural stem cell distribution along the murine ventricular neuraxis may be influenced by cephalic flexures during embryological development6. To date, the rodent brain remains the most popular system in which to study adult neurogenesis. However, the brain of the domestic dog (Canis familiaris), with a size and structural organization intermediate between that of humans and rodents7, represents a valuable yet highly underused animal model. The canine hippocampus in particular embodies this structurally intermediate nature8-10 and can provide a unique perspective on intrinsic variations in neural precursor cell populations. With many closer parallels to the human brain, the canine model may also offer insight into the biology of adult human neurogenesis.
In vitro culture assays have become a key tool for the study of neural precursors and the cellular and biomolecular processes of adult neurogenesis. The neurosphere assay and adherent monolayer culture represent the two predominant systems for expanding neural precursor cells in vitro11-13. Protocols for brain extraction, hippocampal microdissection or neural precursor culture assays have been well documented for the rodent model14-16. However, for the adult canine brain they remain comparatively few17,18, focused instead on fetal or neonatal tissue19-21.
In our published study7 we investigated regional variations in neurogenesis and neural precursor cell populations across the dorsoventral axis of the adult canine hippocampus. Although highly dependent on breed, adulthood in canines is reached between 1 and 3 years of age. Here, we present detailed methods for the extraction, isolation and culture of neural precursor cells from the canine hippocampus. We provide systematic protocols for the expansion of neural precursor cells as both floating neurospheres and as an adherent monolayer culture, and for their subsequent differentiation into mature neural cell types.

<table><tbody><tr><td>1,000 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF1000</td><td></td></tr><tr><td>150 mm Petri dish</td><td>BD Biosciences</td><td>351058</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Greiner Bio One</td><td>188271</td><td></td></tr><tr><td>200 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF200</td><td></td></tr><tr><td>24 well culture plate</td><td>Greiner Bio One</td><td>662160</td><td></td></tr><tr><td>35 mm tissue culture dish</td><td>BD Biosciences</td><td>353001</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>6 well culture plate</td><td>BD Biosciences</td><td>351146</td><td></td></tr><tr><td>B-27 Supplement (50x) serum free</td><td>Life Technologies&amp;nbsp;</td><td>17504044</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td>Life Technologies</td><td>13256029</td><td></td></tr><tr><td>Brain derived neurotrophic factor (BDNF)</td><td>Millipore</td><td>GF029</td><td></td></tr><tr><td>Collagen solution</td><td>Stem Cell Technologies</td><td>04902</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>DMEM (4.5 g/L, D-glucose) 500 ml</td><td>Life Technologies</td><td>11960044</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>BD Biosciences</td><td>354001</td><td></td></tr><tr><td>F-12 nutrient mixture (Ham) (1x) Liquid</td><td>Life Technologies</td><td>31765035</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Life Technologies</td><td>16141079</td><td></td></tr><tr><td>Gelatin from Porcine Skin Type A</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>L-alanyl-L-glutamine dipeptide (GlutaMAX)</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Heparin sodium salt from (porcine)</td><td>Sigma-Aldrich</td><td>H314950KU</td><td></td></tr><tr><td>Laminin (mouse)</td><td>Life Technologies</td><td>23017015</td><td></td></tr><tr><td>NCFC serum free medium (NeuroCult)</td><td>Stem Cell Technologies</td><td>5720</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>Proliferation NS-A (NeuroCult)</td><td>Stem Cell Technologies</td><td>05773</td><td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&amp;nbsp; from Stem Cell Technologies.</td></tr><tr><td>NSC basal medium (Rat; NeuroCult)</td><td>Stem Cell Technologies</td><td>5770</td><td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&amp;nbsp;</td></tr><tr><td>Penicillin/Streptomycin (5,000 U/ml)</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>Povidone-iodine</td><td>Munipharma</td><td>Betadine</td><td></td></tr><tr><td>Trypan blue (0.4%)</td><td>Life Technologies</td><td>15250061</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>Class II biological safety cabinet</td><td>ThermoFisher Scientific</td><td>Safe 2020 1.2</td><td></td></tr><tr><td>Brain knife (disposable)</td><td>Macroknife</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td>ThermoFisher Scientific</td><td>HERAcell 150i</td><td></td></tr><tr><td>Centrifuge</td><td>Hettich</td><td>Universal 320R</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Dumont</td><td></td><td></td></tr><tr><td>Easypet Electric pipette</td><td>Eppindorf&amp;nbsp;</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>Boeco</td><td>Bright-Line Improved Neubauer</td><td></td></tr><tr><td>Manual pipettes</td><td>Eppindorf research</td><td></td><td></td></tr><tr><td>Oscillating bone saw&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Scalpel blades (No.21)</td><td>Paramount</td><td></td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Delta</td><td></td><td></td></tr><tr><td>Water bath</td><td>Grant</td><td>JB Aqua 18 plus</td><td></td></tr></tbody></table>

isolation and expansion of adult canine hippocampal neural precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

Através do uso de precursores neuronais in vitro, ensaios neurogénese e populações de células precursoras neurais foram caracterizadas e comparadas ao longo do eixo dorsoventral do hipocampo adulto canino. células precursoras neurais derivadas de tecido do hipocampo isolado formado neuroesferas flutuante no prazo de 14 dias de isolamento, atingindo um diâmetro de 100 um até 28 dias de cultura. As neuroesferas derivados de dorsal e ventral isolados mostrou nenhuma diferen...

Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

O cérebro canino é um modelo valioso para estudar a neurogênese adulta. Aqui apresentados são protocolos para isolar e expandir adultos células precursoras neurais do hipocampo caninos a partir de tecido cerebral primário.

Isolamento e Expansão da mediana Canino hipocampo Neurais Precursores

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

isolation-and-expansion-of-adult-canine-hippocampal-neural-precursors

Research

JoVE Journal

Developmental Biology

7.0K visualizações.  University of Sydney.  O cérebro canino é um modelo valioso para estudar a neurogênese adulta. Aqui apresentados são protocolos para isolar e expandir adultos células precursoras neurais do hipocampo caninos a partir de tecido cerebral primário. 

Vídeo: Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Crescimento e Diferenciação de Adultos Hippocampal terra ártico Squirrel células-tronco neurais

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Neurociência

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolamento e Expansão do Rato Adulto células-tronco neurais utilizando o ensaio Neurosphere

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Avaliação de dendríticas Arborização no giro dentado do hipocampo em Ratos Região

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

A imuno-histoquímica e Rotulagem múltipla com anticorpos da espécie hospedeira Mesmas para Estudar Adulto Hippocampal Neurogênese

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Seleção de células-tronco neurais de células progenitoras e do rato adulto subventricular Zone e Live-imagem de seus Dynamics com o ciclo celular

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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 ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Regulação específica do circuito de assaying de células precursoras neurais hipocampais adultas

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

Isolamento e Expansão das Neuroesferas a partir de Nichos Neurogênicos de Camundongos (P1-3)

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

<meta charset="utf8"></head><body>Nucleofecção eficiente de NSCs isoladas da zona subventricular murina

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>Autor em destaque: Nucleofecção aprimorada para entrega de genes de alta eficiência em culturas de células-tronco neurais derivadas da zona subventricular murina</

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolamento, expansão e nucleofecção de células-tronco neurais da zona subventricular murina adulta

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...