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>

<ol>
	<li>Kheirbek, M. A., Hen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+vs+ventral+hippocampal+neurogenesis:+implications+for+cognition+and+mood.">Dorsal vs ventral hippocampal neurogenesis: implications for cognition and mood.</a> Neuropharmacol. 36 (1), 373 (2011).</li><li>Tanti, A., Rainer, Q., Minier, F., Surget, A., Belzung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+environmental+regulation+of+neurogenesis+along+the+septo-temporal+axis+of+the+hippocampus.">Differential environmental regulation of neurogenesis along the septo-temporal axis of the hippocampus.</a> Neuropharmacol. 63 (3), 374-384 (2012).</li><li>Eriksson, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+adult+human+hippocampus.">Neurogenesis in the adult human hippocampus.</a> Nat Med. 4 (11), 1313-1317 (1998).</li><li>Bull, N. D., Bartlett, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+mouse+hippocampal+progenitor+is+neurogenic+but+not+a+stem+cell.">The adult mouse hippocampal progenitor is neurogenic but not a stem cell.</a> J Neurosci. 25 (47), 10815-10821 (2005).</li><li>Kempermann, G., 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=Why+and+how+physical+activity+promotes+experience-induced+brain+plasticity.">Why and how physical activity promotes experience-induced brain plasticity.</a> Front Neurosci. 4 (189), 1-9 (2010).</li><li>Golmohammadi, M. G., 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=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26 (4), 979-987 (2008).</li><li>Lowe, A., 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=Neurogenesis+and+precursor+cell+differences+in+the+dorsal+and+ventral+adult+canine+hippocampus.">Neurogenesis and precursor cell differences in the dorsal and ventral adult canine hippocampus.</a> Neurosc. Lett. 593, 107-113 (2015).</li><li>Sasaki, M., 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=MRI+identification+of+dorsal+hippocampus+homologue+in+human+brain.">MRI identification of dorsal hippocampus homologue in human brain.</a> Neuroreport. 15 (14), 2173-2176 (2004).</li><li>Powell, E. W., Hines, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Hippocampus. , 41-59 (1975).</li><li>Jung, M. -. A., 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=Canine+hippocampal+formation+composited+into+three-dimensional+structure+using+MPRAGE.">Canine hippocampal formation composited into three-dimensional structure using MPRAGE.</a> J Vet Med Sci. 72 (7), 853-860 (2010).</li><li>Reynolds, B. 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=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255 (5052), 1707-1710 (1992).</li><li>Ray, J., Raymon, H. K., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+culturing+of+precursor+cells+and+neuroblasts+from+embryonic+and+adult+central+nervous+system.">Generation and culturing of precursor cells and neuroblasts from embryonic and adult central nervous system.</a> Methods Enzymol. 254, 20-37 (1995).</li><li>Oliver-Dela Cruz, J., Ayuso-Sacido, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+from+mammalian+brain:+isolation+protocols+and+maintenance+conditions.">Neural stem cells from mammalian brain: isolation protocols and maintenance conditions.</a> INTECH Open Access Publisher. , (2012).</li><li>Pacey, L., Stead, S., Gleave, J., Tomczyk, K., Doering, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+culture:+neurosphere+generation,+microscopical+analysis+and+cryopreservation.">Neural stem cell culture: neurosphere generation, microscopical analysis and cryopreservation.</a> Nat Protoc. , (2006).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> J Vis Exp. (45), e2393 (2010).</li><li>Walker, T. L., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mouse,+two+cultures:+isolation+and+culture+of+adult+neural+stem+cells+from+the+two+neurogenic+zones+of+individual+mice.">One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice.</a> J Vis Exp. (84), e51225 (2014).</li><li>Lim, J. -. H., Koh, S., Olby, N. J., Piedrahita, J., Mariani, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+neural+progenitor+cells+from+adult+canine+brains.">Isolation and characterization of neural progenitor cells from adult canine brains.</a> Am J Vet Res. 73 (12), 1963-1968 (2012).</li><li>Herranz, C., 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=Spontaneously+Arising+Canine+Glioma+as+a+Potential+Model+for+Human+Glioma.">Spontaneously Arising Canine Glioma as a Potential Model for Human Glioma.</a> J Comp Pathol. 154 (2), 169-179 (2016).</li><li>Walton, R. M., Parmentier, T., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neural+precursor+cell+regions+in+the+rostral+subventricular+zone,+hippocampal+subgranular+zone+and+cerebellum+of+the+dog+(Canis+lupus+familiaris).">Postnatal neural precursor cell regions in the rostral subventricular zone, hippocampal subgranular zone and cerebellum of the dog (Canis lupus familiaris).</a> Histochem Cell Biol. 139 (3), 415-429 (2013).</li><li>Walton, R. M., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+in+neural+progenitor+cells+in+a+dog+model+of+lysosomal+storage+disease.">Abnormalities in neural progenitor cells in a dog model of lysosomal storage disease.</a> J Neuropathol Exp Neurol. 66 (8), 760-769 (2007).</li><li>Milward, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+transplantation+of+multipotential+populations+of+epidermal+growth+factor-responsive,+neural+progenitor+cells+from+the+canine+brain.">Isolation and transplantation of multipotential populations of epidermal growth factor-responsive, neural progenitor cells from the canine brain.</a> J Neurosci Res. 50 (5), 862-871 (1997).</li><li>Ray, J., Peterson, D. A., Schinstine, M., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+differentiation,+and+long-term+culture+of+primary+hippocampal+neurons.">Proliferation, differentiation, and long-term culture of primary hippocampal neurons.</a> Proc. Natl. Acad. Sci. 90 (8), 3602-3606 (1993).</li><li>Palmer, T., Ray, J., Gage, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-2-responsive+neuronal+progenitors+reside+in+proliferative+and+quiescent+regions+of+the+adult+rodent+brain.">FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain.</a> Mol Cell Neurosci. 6 (5), 474-486 (1995).</li><li>Zhu, G., Mehler, M., Mabie, P., Kessler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+progenitor+cell+responsiveness+to+cytokines.">Developmental changes in progenitor cell responsiveness to cytokines.</a> J. Neurosci. Res. 56 (2), 131-145 (1999).</li><li>Whittemore, S. R., Morassutti, D. J., Walters, W. M., Liu, R. -. H., Magnuson, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+and+substrate+differentially+affect+the+lineage+restriction+of+adult+rat+subventricular+zone+neural+precursor+cell+populations.">Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.</a> Exp. Cell. Res. 252 (1), 75-95 (1999).</li><li>Kennedy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+survival+characteristics+of+rat+glial+cells+in+culture.">Postmortem survival characteristics of rat glial cells in culture.</a> J Neurol Neurosurg Psychiatry. 50 (6), 798-800 (1987).</li><li>Viel, J. J., McManus, D. Q., Cady, C., Evans, M. S., Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+and+time+interval+for+culture+of+postmortem+neurons+from+adult+rat+cortex.">Temperature and time interval for culture of postmortem neurons from adult rat cortex.</a> J. Neurosci. Res. 64 (4), 311-321 (2001).</li><li>Huat, T. J., 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=IGF-1+enhances+cell+proliferation+and+survival+during+early+differentiation+of+mesenchymal+stem+cells+to+neural+progenitor-like+cells.">IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells.</a> BMC Neurosci. 15 (91), 1-13 (2014).</li><li>Supeno, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-1+acts+as+controlling+switch+for+long-term+proliferation+and+maintenance+of+EGF/FGF-responsive+striatal+neural+stem+cells.">IGF-1 acts as controlling switch for long-term proliferation and maintenance of EGF/FGF-responsive striatal neural stem cells.</a> Int. J. Med. Sci. 10 (5), 522-531 (2013).</li><li>Ahmed, S., Reynolds, B. 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=BDNF+enhances+the+differentiation+but+not+the+survival+of+CNS+stem+cell-derived+neuronal+precursors.">BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors.</a> J Neurosci. 15 (8), 5765-5778 (1995).</li><li>Wohl, C. 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.

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			<td height="21" style="height:21px;width:337px;">1000 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF1000</td>
			<td style="width:915px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">150 mm petri dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351058</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">15mL centrifuge tubes</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">188271</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">200 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF200</td>
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			<td height="21" style="height:21px;">24 well culture plate</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">662160</td>
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			<td height="21" style="height:21px;">35 mm tissue culture dish</td>
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			<td height="21" style="height:21px;">40 &#181;m cell strainer</td>
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			<td style="width:252px;">351146</td>
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			<td height="21" style="height:21px;width:337px;">B-27 Supplement (50X) serum free</td>
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			<td height="21" style="height:21px;">Basic fibroblast growth factor (bFGF)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">13256029</td>
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			<td height="21" style="height:21px;">Brain derived neurotrophic factor (BDNF)</td>
			<td style="width:196px;">Millipore</td>
			<td style="width:252px;">GF029</td>
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			<td height="21" style="height:21px;">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&#160;</td>
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			<td height="21" style="height:21px;">DMEM (4.5g/L, D-glucose) 500mL</td>
			<td style="width:196px;">Life Technologies&#160;</td>
			<td style="width:252px;">11960044&#160;</td>
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			<td height="21" style="height:21px;width:337px;">DPBS</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">14190250</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Epidermal growth factor (EGF)</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">354001</td>
			<td></td>
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			<td height="21" style="height:21px;">F-12 nutrient mixture (Ham) (1X) Liquid</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">31765035</td>
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			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">16141079</td>
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			<td height="21" style="height:21px;">Gelatin from Porcine Skin Type A</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">G1890</td>
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			<td height="21" style="height:21px;">L-alanyl-L-glutamine dipeptide (GlutaMAX)</td>
			<td>Life Technologies</td>
			<td style="width:252px;">35050061</td>
			<td></td>
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			<td height="21" style="height:21px;">Heparin sodium salt from (porcine)</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">H314950KU</td>
			<td></td>
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			<td height="21" style="height:21px;">Laminin (mouse)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">23017015</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">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&#160;</td>
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			<td height="21" style="height:21px;">Proliferation NS-A (NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">05773</td>
			<td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&#160; from Stem Cell Technologies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NSC basal medium (Rat; NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">5770</td>
			<td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&#160;</td>
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			<td height="21" style="height:21px;width:337px;">Penicillin/Streptomycin (5000 U/mL)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">15070063</td>
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			<td style="width:196px;">Munipharma</td>
			<td style="width:252px;">Betadine</td>
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			<td style="width:196px;">Life Technologies</td>
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			<td style="width:252px;">25200056</td>
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			<td style="width:196px;">ThermoFisher Scientific</td>
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			<td height="21" style="height:21px;width:337px;">Cell culture incubator</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:252px;">HERAcell 150i</td>
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			<td height="21" style="height:21px;width:337px;">Centrifuge</td>
			<td style="width:196px;">Hettich</td>
			<td style="width:252px;">Universal 320R</td>
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			<td height="21" style="height:21px;width:337px;">Manual pipettes</td>
			<td style="width:196px;">Eppindorf research</td>
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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

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolamento de Neural Stem / células progenitoras da Região Periventricular do rato adulto e Medula Espinhal Humano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Biologia do Desenvolvimento

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Estável e eficiente modificação genética de células do rato adulto V-SVZ para a Análise da Neural Stem Cell Autónoma e Efeitos não autónomos

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Ex Cultura Vivo de Pintainho Cerebelar Slices e voltadas para o espaço Eletroporação do grânulo precursores de células

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Isolamento e Cultura de neuroesferas Zebrafish Adulto derivado do cérebro

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Cultura pluripotentes humanas e células-tronco neurais em um sistema de cultura celular fechado de Pesquisa Básica e pré-clínica

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Isolamento e expansão de CTM / células estromais derivadas de placenta humana Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Estimulação da sinalização Notch no mouse osteoclastos Precursores

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

O isolamento e a cultura de células primárias de Epicárdica derivado de espécimes de coração adulto e Fetal humano

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Derivação e diferenciação de células-tronco mesenquimais ovário canina

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Citometria de fluxo de viver-pilha em tempo real para investigar o influxo de cálcio, formação de poros e fagocitose pelos receptores P2X7 em células progenitoras neurais adulto

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...