Esta pesquisa foi apoiada pelo Programa de Pesquisa intramuros no National Institutes of Health, NINDS. Os autores gostariam de agradecer a todos os membros do Laboratório de Medicina Molecular e Neurociência, Rick Dreyfuss para ajudar com microscopia e peneiramento Pamela C. para ajudar com a edição.

Nota: Para a cultura de rotina de células progenitoras neurais e células da linhagem oligodendrocytic, a incubação é realizada a 37 ° C em 5% de CO 2 humidificado atmosfera. Cada 2 dias, o meio é substituído com 50 a 100% de meio fresco se a cultura é de 40-70% confluentes. No momento da confluência perto, as culturas são passadas a 2-2.5e6/T75 frasco geralmente num plano semanal. 1. Preparando o balão revestido <ol><li> Para preparar frascos revestidos diluir 5 mg de poli-D-lisina (PDL) em 100 ml de água desionizada (DI-água), em seguida, casaco frascos T75 com 50 ug / ml de PDL à temperatura ambiente (TA) no escuro. Após 1 ½ hora, aspirar o PDL e lavar os frascos de uma vez com DI-água. Deixe os frascos secos antes de semear as células (Tabela 1). </li></ol> 2. Começando diferenciação das células progenitoras neurais em oligodendrócitos Progenitoras derivados (DOP) O protocolo para seolate células progenitoras do SNC é descrito numa publicação anterior e não faz parte do presente protocolo 1. As células progenitoras do SNC, foram isolados do telencéfalo de um cérebro de 8 semanas de gestação fetal, obtidas em conformidade com as directrizes de NIH. <ol><li> Crescer população multipotencial das células progenitoras neurais em PDL frascos revestidos em meio neurobasal suplementado com 25 ng / ml de bFGF e 20 ng / ml do factor de crescimento epidérmico (EGF) (Figura 1A). Não usá-los se for maior do que a passagem 11 (p11). </li><li> Começam a diferenciar as células progenitoras neuronais, quando a cultura é de aproximadamente 70% confluentes. </li><li> Fazer meio de diferenciação completa oligodendrócitos usando DMEM livre de soro / HAMS F12 01:01 suplementado com albumina de soro bovino, L-glutamina, gentamicina, componentes N2, T3, Shh, NT-3, bFGF, e PDGF-AA (Tabela 1) . Este meio será definida como o meio de oligo com factores de crescimento (meio de oligo + GF). </li><li> Remover PROGENItor media dos frascos, lave as células uma vez com salino tamponado com fosfato (PBS), em seguida, adicionar meio de oligo + GF (detalhes descritos em 2.3) para iniciar a diferenciação oligodendrocítica. Apenas as células comprometidas com a linhagem oligodendrocytic crescerá (Figura 1B). Cultura das células neste meio durante 3 semanas. </li><li> Cada semana, quando as células estão a 90-95% de confluência passagem, eles utilizando tripsina (0,05%), EDTA (0,1%) (4 ml para um frasco T75). Transferir todo o meio a partir das células para ser passado para um tubo cónico de 50 ml, o meio condicionado será utilizado para temperar a tripsina. Certifique-se de não secar as células. Em seguida, adicionar tripsina. </li><li> Incubar as células em tripsina durante 5 min à TA, batendo suavemente o frasco várias vezes para ajudar destacamento celular. </li><li> Extingue-se a tripsina por adição de 4-5 ml de meio condicionado para o balão com as células isoladas. </li><li> Transferir o meio e as células para o mesmo tubo cónico de 50 ml. Centrifuga-se o meio e as células a 1200 rpm (~300 xg) durante 5 min à temperatura ambiente. </li><li> Aspirar o sobrenadante, ressuspender o sedimento de células, inclinando gentilmente o tubo e, em seguida, adicionar meio fresco oligo + GF. Gentilmente pipeta meio e as células para cima e para baixo para se obter uma suspensão de célula uniforme. </li><li> Contar as células e 2.5e6 de transferência em cada novo PDL revestido T75 frasco. </li></ol> 3. Etapa final da diferenciação de oligodendrócitos <ol><li> Após 3 semanas de cultura em meio de oligo + GF, quando as células estão a 70-80% de confluência, iniciar o processo final de diferenciação (Figura 1C). </li><li> Preparar meio da mesma maneira como o meio de oligo + GF mas sem adicionar o GF quatro: Shh, NT-3, bFGF e PDGF-AA. Este meio sem o GF quatro serão definidos como oligo meio - GF. </li><li> Aspirar o meio de oligo + GF do frasco, lavar uma vez com PBS e, em seguida, adicionar um volume total de 16 ml de meio, que de ¾ (12 ml) é oligo meio - GF e ¼ (4 ml) é PDN meio condicionado, por 6-10 Dias). O uso de meio condicionado a partir de PDN não é crítico para o processo de diferenciação ou a sobrevivência das células. Nós, de facto, observou-se que só o contacto directo com as células de DOP PDN em experiências de co-cultura tinha um efeito sobre a duração da sobrevivência DOP. O tempo de sobrevivência médio em oligo - GF pode ser prolongada até duas ou três semanas, se as células de DOP são co-cultivados com células PDN. O protocolo para diferenciar as células progenitoras neurais em PDN é descrito numa publicação anterior e não faz parte do presente protocolo 1. Retirada do factor de crescimento a partir dos resultados de cultura de média em cultura progressivamente diferenciadas de células com vários processos (Figura 1D). </li></ol> 4. Ensaio de Citometria de Fluxo O ensaio de citometria de fluxo compara a aquisição de marcadores de oligodendrócitos, durante o processo de diferenciação, em relação com as da sua população parental. <ol><li> Use pró neuralgenitores como controle. Semente de células DOP 2.5x10 6 células em dois frascos T75 revestidos com poli-D-lisina em meio de oligo + GF. </li><li> Uma semana mais tarde, substitua meio com meio fresco oligo + GF em frascos como uma cultura de controlo para a viabilidade celular, enquanto que outros frascos tinham oligo meio - GF. </li><li> Para dissociar as células em ambos os meios DOP usar 20 U / ml de papaína e não tripsina. Isto é porque a papaína tem um suave efeito sobre as células durante a dissociação preservação da morfologia celular. </li><li> Adicionar 5 ml de solução salina equilibrada de Earle (EBSS) para um frasco de papaína. Colocar o frasco a 37 ° C durante 10 minutos ou até que a papaína esteja completamente dissolvido e a solução é mais clara. Esta é a solução de papaína utilizada para dissociar as células. </li><li> Adicionar 0,5 mL de EBSS para um frasco de desoxirribonuclease I (DNase) (Tabela 1). Misture delicadamente. Adicionar 0,25 ml desta solução para o frasco de papaína dissolvida. Esta preparação contém uma concentração final de aproximadamente 20 U / ml de papaina, e 00,005% de DNase. </li><li> Solução de papaína lugar (como descrito acima), em que as células e incubar a 37 ° C durante 15-30 min; monitorar o desprendimento das células utilizando um microscópio. Parar a reacção com 5 ml de meio com ou sem GF e centrifugar a 1200 rpm durante 7 min. </li><li> Transferir os sedimentos celulares a partir de ambos os balões em dois diferentes tubos de 5 ml de polipropileno e lavá-las com meio fisiológico normal (NPM: 145 mM de NaCl, 5 mM de KCl, 1,8 mM CaCl2, 10 mM de Hepes e 10 mM de glucose) suplementado com 1 mg / ml albumina de soro bovino (BSA + NPM). </li><li> Executar superfície imunocoloração a 4 ° C durante 20 minutos utilizando específicos e apropriadamente titulado anticorpos primários para A2B5, O4, GalC (Tabela 2). </li><li> Lavar as células com NPM + BSA a 4 ° C durante 5 min e incube-as com anticorpos específicos secundários, a 4 ° C durante 20 minutos (Tabela 2). </li><li> Para corar os antigénios intracelulares, incluindo nestina, proteína glial fibrilar ácida (GFAP), classe III e serta;-tubulina e a proteína básica mielina (MBP) (Tabela 2), as células fixar em 2% de paraformaldeído (PFA), durante 20 min à TA e permeabilizar com etanol a 70% frio durante 15 min a -20 ° C. </li><li> Lavar as células com PBS 1x e incubar com anticorpos específicos secundários (Quadro 2). </li><li> Para discriminar entre células intactas e fragmentos subcelulares, durante a análise de citometria de fluxo, corar as células com 4,6-diamidino-2-phenyllindole, o dicloridrato de (DAPI) (Life Technologies, Grand Island, NY). </li><li> Na otimização do protocolo acima, foram realizados todos os experimentos de controle apropriados para confirmar a especificidade de cada imunorreagente. Resumidamente, para os anticorpos marcados directamente, o controlo negativo adequado era um anticorpo conjugado directamente da mesma classe de imunoglobulinas do isotipo (ou subclasse) e conjugado de fluorocromo como o anticorpo primário (isto é, o controlo de isotipo). Para imunomarcação indireta de anticorpos primários, o adequado controle negativo wcomo anticorpo secundário conjugado com o fluorocromo de interesse que especificamente dirigida a classe de imunoglobulina (ou subclasse) do hospedeiro, em que o anticorpo primário foi gerado. Quando os anticorpos primários a partir de hospedeiros múltiplos (rato, coelho e galinha) foram utilizados em conjunto, utilizou-se o anticorpo secundário de segmentação do anticorpo primário de cada máquina e cada um anticorpo secundário usado foi tipicamente cruzada adsorvido contra as imunoglobulinas de outros hospedeiros para minimizar transversal imunorreatividade hospedar. Os controlos positivos incluíram imunocoloração directa ou indirecta de preparações de células que se sabe expressarem os antigénios de interesse utilizando quer imunorreacções, directas ou indirectas, com os anticorpos primários específicos para estes antigénios. Os métodos de optimização acima foram efectuados uma vez para cada tipo de imunocoloração experimentos. Imunorreacções de controlo não revelou imunorreactividade cruzada epítopo significativa entre os anticorpos primários e secundários. A citometria de fluxo foi realizada de forma uniformesuspensão de células marcadas por fluorescência utilizando um classificador FACVantage célula SE (BD Biosciences, San Jose, CA) equipado com três lasers, que proporcionam comprimentos de onda de excitação de 488 nm sintonizados, 647 nm, e uma gama de UV (351-364 nm) (Figura 2) . </li></ol> 5. Ensaio de Imunofluorescência Nota: Para as experiências de imunofluorescência, DOP são plaqueadas em meio de oligo + GF ou meio de oligo - GF em 2.5e5 em placas de 6 poços revestidas com PDL. As células são fixadas em 2% de PFA em vários pontos de tempo após a retirada de factores de crescimento. O anticorpo mancha é realizada em placas de 6 cavidades de plástico em vez de lamelas de vidro. <ol><li> Remova a mídia e adicione o PFA 2%. Incubar durante 10 min à TA. Descarte PFA. Lavar as células 3 vezes com PBS, 5 minutos de cada vez. Tenha cuidado para não secar as células. </li><li> Permeabilizar as células para marcadores intracelulares utilizando solução de triton 0,25% em PBS durante 10 min à RT. </li><li> Para evitar ligação não específicos de células, bloco fou 10 min com HHG (1 mM de tampão HEPES, soro de cavalo a 2% e soro de cabra a 10% em solução salina equilibrada de Hanks). Diluir anticorpos específicos primários (Tabela 2) até uma concentração pré-determinada em HHG. Coloque anticorpos diluídos em células. Incubar 1 hora à temperatura ambiente no agitador ou a 4 ° C durante a noite. </li><li> Lavar as células 3 vezes com PBS, durante 5 minutos de cada vez. Diluir anticorpos secundários apropriados (Tabela 2), acoplado com um fluorocromo a uma concentração pré-determinada em HHG. Coloque a mistura de anticorpos secundários diluídos em células. Incubar 1 hora à temperatura ambiente num agitador no escuro. </li><li> Lavar as células 3 vezes com PBS, durante 5 minutos de cada vez. </li><li> Para evitar a fotodegradação adicionar 10 ul de Prolong ouro com DAPI para as células e colocar uma lamela de vidro em cima deles. Visualizar as células marcadas, utilizando um microscópio de fluorescência Axiovert 200M (Zeiss, Thornwood, NY) (Figura 3). </li></ol>

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Este protocolo descreve como para derivar a partir de oligodendrócitos fetais humanas primárias células progenitoras neuronais e caracterizar o fenótipo de citometria de fluxo utilizando ambos e coloração por imunofluorescência. A expansão eo crescimento de células progenitoras neurais de SNC fetal foi muito bem descrito 1-4. No entanto, a obtenção de oligodendrócitos humanos suficientes para experimentação in vitro continua a ser difícil, embora seja possível identificar e i...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Concentração final </td></tr><tr><td> DME / PRESUNTOS F12 01:01 </td><td> Omega Cientista </td><td> DM-251 </td><td> 1X </td></tr><tr><td> Bovine Albumin </td><td> Sigma </td><td> A9418 </td><td> 1% </td></tr><tr><td> Gentamicina </td><td> Qualidade Biologicals </td><td> 120-098-031 </td><td> 50 ug / ml </td></tr><tr><td> L-Glutamina </td><td> Qualidade Biologicals </td><td> 118-084-061 </td><td> 2 mM </td></tr><tr><td> T3 </td><td> Sigma </td><td> T2877 </td><td> 3 nM </td></tr><tr><td> Componentes N2 </td><td> Gibco BRL </td><td> 17502 </td><td> 1:100 </td></tr><tr><td> NT-3 </td><td> PeproTech Inc </td><td> 450-03 </td><td> 2 ng / ml </td></tr><tr><td> Shh </td><td> R &amp; D System </td><td> 1314-SH/CF </td><td> 2 ng / ml </td></tr><tr><td> bFGF </td><td> PeproTech Inc </td><td> 100-18B </td><td> 20 ng / ml </td></tr><tr><td> PDGF-AA </td><td> PeproTech Inc </td><td> 100-13A </td><td> 10 ng / ml </td></tr><tr><td> PDL </td><td> Sigma </td><td> P6407 </td><td> 50 ^ g / m </td></tr><tr><td> PFA </td><td> Electron Microscopy Sciences </td><td> 15712 </td><td> 2% </td></tr><tr><td> Tripsina </td><td> Qualidade Biologicals </td><td> 118-087-721 </td><td></td></tr><tr><td> Papaína </td><td> Worthington </td><td> LK003178 </td><td> 20 U / ml </td></tr><tr><td> DNase frascos </td><td> Worthington </td><td> LK003172 </td><td> 0,005% </td></tr><tr><td> EBSS </td><td> Worthington </td><td> LK003188 </td><td></td></tr><tr><td> Prolongar Ouro comDAPI </td><td> Invitrogen </td><td> P36931 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabela 1. Reagentes. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Primário Abs (todos a 1 ug / ml) </td><td></td><td> Isotype espécies </td><td> Fonte </td><td> Abs secundário </td></tr><tr><td colspan="5"> Citometria de Fluxo </td></tr><tr><td> A2B5-Biotina </td><td> Mouse </td><td> IgM </td><td> Dom J. Nielson </td><td> Estreptavidina PETR, Invitrogen, CA </td></tr><tr><td> O4FITC </td><td> Mouse </td><td> IgM </td><td> Presente de J. Nielson </td><td></td></tr><tr><td> A2B5 </td><td> Mouse </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-FITC, Invitrogen, CA </td></tr><tr><td> O4 </td><td> Mouse </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-FITC, Invitrogen, CA </td></tr><tr><td> GalC </td><td> Mouse </td><td> IgG 3 </td><td> Millipore, MA </td><td> gαm IgG 3-PE, Southern Biotech, AL </td></tr><tr><td> MBP </td><td> Frango </td><td> IgY </td><td> Millipore, MA </td><td> dαck IgY-AMCA, Jackson Immu., PA </td></tr><tr><td> Nestina </td><td> Mouse </td><td> IgG 1 </td><td> Messam et al. 2000 28 </td><td> gαm IgG 1-PECy5, Invitrogen, CA </td></tr><tr><td> GFAP </td><td> Coelho </td><td> IgG </td><td> Millipore, MA </td><td> gαrb IgG-PE, Jackson Immu, PA </td></tr><tr><td> tubulina βIII </td><td> Mouse </td><td> IgG2 </td><td> Covance, CA </td><td> gαm IgG-2-PETR, Invitrogen, CA </td></tr><tr><td colspan="6"> Imunocitoquímica </td></tr><tr><td> tubulina βIII (1:1,500) </td><td> Mouse </td><td> IgG 2a </td><td> Covance, CA </td><td> gαm IgG 2a-FITC, Invitrogen, CA (1:1,000) </td></tr><tr><td> GFAP (1:1,000) </td><td> Coelho </td><td> IgG </td><td> Millipore, MA </td><td> gαrb IgG-FITC, Jackson Immu, PA (1:500) </td></tr><tr><td> MBP (1:50) </td><td> Frango </td><td> IgY </td><td> Millipore, MA </td><td> dαck IgY-FITC, Jackson Immu, PA (1:100) </td></tr><tr><td> O4 (1:100) </td><td> Mouse </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-AF546, Invitrogen, CA (1: 100) </td></tr><tr><td> GalC (1:10) </td><td> Coelho </td><td> IgG </td><td> Millipore, MA </td><td> gαm IgM-AF750, Invitrogen, CA (1:100) </td></tr></tbody></table> . Tabela 2 Os anticorpos (Abs) utilizados para ensaios de citometria de fluxo e imunocitoquímica conjugados de anticorpos: PE, ficoeritrina; PETR, ficoeritrina Texas Red; AMCA, amino-metil-cumarina-acetato; Cy, cianina; FITC, isotiocianato de fluoresceína de Ig:. Imunoglobulinas. . AF: Alexa Fluor; gαm: cabra anti-rato; gαrb: cabra anti-coelho; dαck: burro anti-galinha. </td></tr></tbody></table>

progenitor-derived oligodendrocyte culture system from human fetal brain

Diferenciação de progenitores neurais humanas em tipos de células neuronais e gliais oferece um modelo para estudar e compará-regulação molecular do desenvolvimento neural de células de linhagem. Expansão in vitro de células progenitoras neurais a partir de tecido fetal CNS tem sido bem caracterizado. Apesar de a identificação e isolamento de células progenitoras da glia do adulto assunto sub-cortical humano branco e desenvolvimento de várias condições de cultura de diferenciação directa de progenitores neurais fetais em oligodendrócitos de mielina que produzem, adquirindo suficientes oligodendrócitos humanos para experimentação in vitro continua a ser difícil. Diferenciação de galactocerebrósido + (GalC) e O4 + precursor oligodendrócitos ou células progenitoras (OPC) a partir de células precursoras neurais tem sido relatado o uso de segundo trimestre cérebro fetal. No entanto, estas células não proliferam na ausência de células de suporte, incluindo os astrócitos e neurónios, e são rapidamente perdidos ao longo do tempo na cultura. Permanece a necessidade para um sistema de cultura para a produção de células da linhagem de oligodendrócitos adequado para experimentação in vitro. Cultura de oligodendrócitos humanos primários poderiam, por exemplo, ser um modelo útil para estudar a patogénese da neurotrópicas agentes infecciosos, como o poliomavírus humanos, JCV, que in vivo infecta as células. Estas culturas de células podem também proporcionar modelos de outras doenças desmielinizantes do sistema nervoso central (SNC). Primária, fetal humano derivado do cérebro, as células progenitoras neurais multipotenciais proliferam in vitro, mantendo a capacidade de se diferenciar em neurônios (células progenitoras derivadas de neurônios, PDN) e astrócitos (células progenitoras derivadas de astrócitos, PDA) Este estudo mostra que progenitores neurais pode ser induzida para diferenciar através de muitos dos estágios de desenvolvimento da linhagem oligodendrocytic (progenitor derivados de oligodendrócitos, DOP). Nós, de cultura de células progenitoras neurais em meio isento de soro F12 de DMEM-supcomplementado com um factor de crescimento de fibroblastos básico (bFGF), factor de crescimento derivado de plaquetas (PDGF-AA), Sonic hedgehog (Shh), factor neurotrófico 3 (NT-3), N-2 e tri-iodotironina (T3). As células cultivadas são passadas a células 2.5e6 por frascos de 75 centímetros aproximadamente de sete em sete dias. Usando estas condições, a maioria das células na cultura de manter uma morfologia caracterizada por alguns processos e marcadores expressos do pré-oligodendrócitos células, tais como A2B5 e O-4. Quando se remover os factores de crescimento de quatro (GF) (bFGF, PDGF-AA, Shh, NT-3) e adicionar o meio condicionado a partir de PDN, as células começam a adquirir mais processos e os marcadores de diferenciação específicos expressos oligodendrócito, tais como GalC e mielina proteína básica (MBP). Foi realizada a caracterização fenotípica usando multicolor citometria de fluxo para identificar marcadores únicos de oligodendrócitos.

É muito importante para iniciar o processo de diferenciação de uma cultura de células de 70% -80% confluente neural progenitor (Figura 1A). Muitas células irão morrer depois de mudar o meio de cultura de células progenitoras para médio oligo uma vez que inclui fatores de crescimento específicos. Isto indica que o crescimento de células progenitoras neurais não comprometidos com um fenótipo oligodendrocytic não serão suportadas pelo meio novo (Figura 1B). A incubação em m...

Watch this Scientific Journal Video about Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain at JoVE.com

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Primário, fetais humanos derivado do cérebro, as células progenitoras multipotenciais proliferam<em&gt; In vitro</em&gt; Enquanto se mantém a capacidade de se diferenciar em neurónios e astrócitos. Este trabalho mostra que os progenitores neurais podem ser induzidas a diferenciar por estágios da linhagem oligodendrocytic por condicionamento com factores de crescimento seleccionados.

Progenitor derivado Sistema de Cultura oligodendroglial Cérebro Fetal Humano

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

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Research

JoVE Journal

Neuroscience

14.8K visualizações.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. Primário, fetais humanos derivado do cérebro, as células progenitoras multipotenciais proliferam In vitro Enquanto se mantém a capacidade de se diferenciar em neurónios e astrócitos. Este trabalho mostra que os progenitores neurais podem ser induzidas a diferenciar por estágios da linhagem oligodendrocytic por condicionamento com factores de crescimento seleccionados.

Vídeo: Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

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

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Neurociência

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

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

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

Isolamento e cultura de células endoteliais da Embryonic posencéfalo

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

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

Um Método de Expansão cGMP-aplicável para Agregados de Neural Humano-tronco e células progenitoras derivadas de células-tronco pluripotentes ou tecido cerebral do feto

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

Viver a imagem das células do córtex Cerebral primário usando um sistema de cultura 2D

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Simples geração de uma cultura de alto rendimento de neurônios induzidas de fibroblastos de pele humana adulta

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has provided important insights into fundamental processes of the brain like cognition, learning, and motor control. However, this approach cannot provide causal evidence for the involvement of brain areas of interest. Transcranial magnetic stimulation (TMS) is a powerful, noninvasive tool for studying the human brain that can overcome this limitation by transiently modifying brain activity. Here, we highlight recent advances using a paired-pulse, dual-site TMS method with two coils that causally probes cortico-cortical interactions in the human motor system during different task contexts. Additionally, we describe a dual-site TMS protocol based on cortical paired associative stimulation (cPAS) that transiently enhances synaptic efficiency in two interconnected brain areas by applying repeated pairs of cortical stimuli with two coils. These methods can provide a better understanding of the mechanisms underlying cognitive-motor function as well as a new perspective on manipulating specific neural pathways in a targeted fashion to modulate brain circuits and improve behavior. This approach may prove to be an effective tool to develop more sophisticated models of brain-behavior relations and improve diagnosis and treatment of many neurological and psychiatric disorders.

This article describes new approaches to measure and strengthen functionally specific neural pathways with transcranial magnetic stimulation. These advanced noninvasive brain stimulation methodologies can provide new opportunities for the understanding of brain-behavior relations and development of new therapies to treat brain disorders.

Medir e manipular vias neurais funcionalmente específicas no sistema motor humano com estimulação magnética transcraniana

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

Geração de Oligodendrocytes e Oligodendrocyte-Conditioned Medium para Experimentos de Cocultura

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissecção e isolamento da glia murina de múltiplas regiões do sistema nervoso central

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Obtenção de Microglia Humana de Tecido Cerebral Humano Adulto

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

Geração de neurônios humanos e oligodendrócitos a partir de células-tronco pluripotentes para modelagem de interações neurônio-oligodendrócitos

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...