Este trabalho foi financiado pelo financiamento da Fundação Overstreet.

1. Configuração básica antes de prosseguir para dissecção: <ol><li> Volume apropriado de meio NSC completo é preparado pela mistura NeuroCult NSC Basal Medium e Suplementos NeuroCult NSC Proliferação na proporção 9:1, respectivamente. </li><li> O meio é aquecido em banho-maria a 37 ° C. </li><li> Frio HEPES-buffered meio mínimo essencial (HEM), com alta concentração de antibióticos (10%) está preparado para dissecção e propósito de lavar roupa. Alternativamente, NSC meio basal com a suplementação de antibióticos também pode ser utilizado para esta finalidade. </li><li> 25-30 mL de HEM frio contendo antibióticos é dispensada em tubos de 50 ml estéril para a coleta dos embriões. </li><li> Vários pratos de plástico Petri 10 centímetros são necessários para manter os embriões e os cérebros durante a dissecção e também para manter o tecido dissecado. </li><li> Os instrumentos cirúrgicos, necessários para remover os embriões (tesouras grandes, pequenas tesouras pontudas, pinças grandes, pequenas pinças curvas) ou para a dissecção do cérebro embrionário (pequeno fórceps, pinças curvas finas, em ângulo de 45 ° pinças finas e pequenas tesouras) são esterilizados usando conta de vidro estufa a 250 ° C ou outros métodos disponíveis autoclave. </li><li> Microscópio de dissecção é limpa com álcool 70% e criar dentro de um fluxo laminar ou capuz PC2. </li></ol> 2. Colheita E14 Cérebro de Rato e Micro-dissecção: <ol><li> Uma vez acoplado rato grávida é anestesiado no dia 14 de gestação de acordo com um protocolo de animais institucional aprovado. </li><li> Deslocamento cervical é realizada para se certificar de que o animal não sofre dor e angústia. </li><li> O rato anestesiado é colocado em sua parte traseira em um lenço de papel absorvente, eo abdômen é lavado com etanol 70% para esterilizar a área. </li><li> A pele sobre o abdômen é apreendido com uma pinça grande, e, em seguida, a pele ea fáscia é cortado com uma tesoura grande para expor a cavidade abdominal e os cornos uterinos. </li><li> O útero são removidos com uma pinça e tesouras pequenas e são transferidos para um tubo contendo 50 ml cônico HEM frio. </li><li> Os tecidos uterinos são transferidos para a capa e depois lavados uma ou duas vezes com volume suficiente de HEM estéril limpa e fria para remover possíveis contaminantes como o sangue e cabelo. </li><li> Os tecidos uterinos são então transferidos para um prato de Petri contendo 10 centímetros HEM frio. </li><li> Os cornos uterinos são abertos usando pequenas pinças e tesouras curvas e os embriões são transferidos para um prato de 10 centímetros novo, que contém HEM frio. </li><li> As cabeças dos embriões são separados ao nível da medula espinhal cervical e transferidos para outra placa de Petri contendo HEM frio. </li><li> Placa de Petri é transferida sob um microscópio de dissecação para remover o cérebro do crânio. </li><li> As cabeças são realizadas com uma pinça fina curva do lado caudal assim como o lado dorsal da cabeça são voltados para cima. Usando micro-tesouras, um primeiro corte horizontal é feita acima dos olhos e, em seguida, continuou na linha mediana da testa para a parte traseira da cabeça. Certifique-se de cortar a pele eo crânio e para não danificar o cérebro subjacente. </li><li> O cérebro é retirado do crânio, pressionando as bordas da seção de corte em um movimento para trás usando a pinça curva. Este procedimento continua até que todos os cérebros são removidos os crânios. </li><li> O cérebro é mantida constante utilizando a pinça curva de modo que o lado dorsal é voltada para cima e em seguida, usando microtesoura um corte é realizado através do córtex de cada hemisfério se estende do bulbo olfatório à parte traseira do hemisfério para expor as eminências ganglionares. </li><li> Usando a pinça curva em uma mão e as pinças de 45 ° angulada no outro, as abas de corte de córtices estão espalhados e as eminências ganglionares são dissecados. </li><li> O tecido dissecado é colocado em um prato de 10 centímetros de Petri estéreis, e este procedimento é repetido até que todos os cérebros foram micro-dissecados. </li><li> Pedaços de tecidos são coletadas utilizando 1 mL de meio de NSC em uma ponta da pipeta 1 mL e transferido para um tubo de centrifugação de 15 mL. </li><li> Tecido é então dissociado completamente, mas com cuidado, pressionando a ponta da pipeta no fundo do tubo e pipetar a suspensão para cima e para baixo. Desta forma, os aglomerados são quebrados em uma única célula. Pipetagem é realizado 3-4 vezes para que uma suspensão leitosa como é alcançado. Então, a suspensão é deixada em repouso por 1-2 minutos, de modo a clumps não dissociada precipitados. </li><li> Quase a suspensão célula inteira é transferida para outro tubo e depois outro de 1 mL de meio de NSC é adicionado ao clumps restantes e dissociado a única célula, como descrito. </li><li> O conteúdo dos tubos são reunidos e centrifugados por 5 minutos em temperatura ambiente, a 700rpm (110g). </li><li> O sobrenadante é aspirado off até o pellet real, e as células são ressuspendidas em 1 mL de meio de NSC completa. </li><li> O meio é gentilmente pipetado cima e para baixotem uma suspensão celular homogênea único. </li><li> 10 L da suspensão de células é misturado com 90 mL de azul de tripano para realizar uma contagem de células. </li><li> Finalmente, as células são banhados na densidade de 2x10 5 células / mL em meio NSC completo suplementado com 20ng / mL fator de crescimento epidérmico (EGF). 5 mL médio é usado para T25, T75 para 20 mL e 40 mL para frascos T175. </li><li> Neurospheres são formados em 5-7 dias, quando incubados a 37 ° C em uma incubadora umidificada com 5% de CO 2. Em 6-7 dias, as esferas devem medir entre 150-200 microns e estará pronto para subcultura (passagem). </li></ol> 3. Passaging e Expansão da NSCs embrionárias: <ol><li> Quando o neurospheres estão prontos para subcultura (150-200 m de diâmetro), o meio com esferas suspensão é removido dos frascos, colocado em um tubo de cultura apropriada estéril tamanho do tecido, e centrifugado a 700 rpm (110 g) por 5 min em temperatura ambiente. </li><li> O sobrenadante é descartado e as esferas são suspensas em 1 mL de 0,05% de tripsina-EDTA. </li><li> A suspensão de células é, então, incubadas em banho-maria a 37 ° C por 2-3 min, em seguida, um volume igual de inibidor de tripsina de soja é usado para interromper a atividade de tripsina. </li><li> A suspensão de células é suavemente pipetado cima e para baixo para garantir que a tripsina foi completamente inativada. </li><li> A suspensão celular é centrifugado a 700 rpm (110g) por 5 min. Então, o sobrenadante é removido e as células são ressuspendidas em 1 mL de meio de NSC completa. </li><li> 10 L da suspensão de células é misturado com 90 mL de azul de tripano para realizar uma contagem de células. </li><li> As células são banhados em uma concentração de 5x10 4 células / mL em meio NSC completo suplementado com 20ng / mL EGF em um frasco apropriado cultura dimensão do tecido. 5 mL médio é usado para T25, T75 para 20 mL e 40 mL para frascos T175. </li><li> Neurospheres secundário são formados em 5-7 dias, quando incubados a 37 ° C em uma incubadora umidificada com 5% de CO 2. </li></ol> 4. Resultados representativos: No ensino primário cultura NSC embrionárias, a maioria das células vai se tornar hipertrófica e anexar aos pratos de cultura de tecidos em placas. Embora a maioria das células vão morrer ou diferenciar, depois de 2-3 dias, as células proliferativas fazer pequenos aglomerados de células que irá separar a partir do substrato (Figura 1). Formação de grandes agregados esferoidais nas primeiras 48 horas de cultura não deve ser confundida com esferas primárias. Formação de agregados depende principalmente da quantidade de detritos e pedaços de tecido não-dissociada da cultura. Neurospheres verdadeiros são os fase brilhante e tornar-se mais esférico à medida que aumenta de tamanho (Figura 2). Microspikes pequena aparece na superfície externa das esferas viável e saudável (Figura 3). Depois de 5-7 dias, as esferas devem ser redondos, mas não compactado, e deve medir entre 150 e 200 m de diâmetro. Se neurospheres são permitidos a crescer muito grande (após 90-10 dias em cultura), eles podem formar agregados ou tornar-se de cor escura por causa da morte de células no centro das esferas (ver vídeo). Neurospheres grande poderia, eventualmente, começam a se diferenciar in situ (fixação ao substrato e migrando para a periferia). Também é difícil dissociar neurospheres grande e subcultura-los. <img src="/files/ftp_upload/2457/2457fig1.jpg" alt="Figura 1" /> Figura 1. Primário E14 cultura NSC três dias após o plaqueamento. Setas mostram a proliferação de agrupamentos NSCs. Ampliação original; 20x <img src="/files/ftp_upload/2457/2457fig2.jpg" alt="Figura 2" /> Figura 2. Primária E14 cultura NSC 7 dias após o plaqueamento. Ampliação original; 10x <img src="/files/ftp_upload/2457/2457fig3.jpg" alt="Figura 3" /> Figura 3. Passage um E14 neurospheres cinco dias após o plaqueamento. Observe a picos de micro-(setas) na periferia das esferas. Ampliação original; 20x

<ol>
	<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, 1707-1710 (1992).</li><li>Morshead, C. M. <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+in+the+adult+mammalian+forebrain:+a+relatively+quiescent+subpopulation+of+subependymal+cells.">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> Neuron. 13, 1071-1082 (1994).</li><li>Craig, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+growth+factor+expansion+of+endogenous+subependymal+neural+precursor+cell+populations+in+the+adult+mouse+brain.">In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.</a> J Neurosci. 16, 2649-2658 (1996).</li><li>Golmohammadi, M. G. <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, 979-987 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., BA, R. e. y. n. o. l. 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=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. , (2010).</li><li>Reynolds, B. A., Rietze, R. 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+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+neural+stem+and+progenitor+cells+in+the+neural+colony-forming+cell+assay.">Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.</a> Stem Cells. 26, 988-996 (2008).</li></ol>

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O ensaio neurosphere é o método de escolha para o isolamento e expansão de células-tronco neurais 05/01 devido à sua simplicidade e reprodutibilidade. Este ensaio é uma ferramenta inestimável para a geração em larga escala de células indiferenciadas células do SNC precursor, o que poderia ser usado para ambos in vitro e in vivo. Deve ser enfatizado que neurospheres poderiam ser gerados a partir de ambos os bona fide células-tronco neurais e progenitores mais restrito. Portanto, o...

* Para fazer HEM, misture 110L pacote de MEM and160ml de HEPES 1M e obter um volume de 8,75 L com água destilada. Definir o pH final para 7,4 e armazená-lo a 4 ° C.

establishing embryonic mouse neural stem cell culture using the neurosphere assay

Em mamíferos, as células-tronco atua como uma fonte de células indiferenciadas para manter gênese ea renovação celular em diferentes tecidos e órgãos, durante o tempo de vida do animal. Eles podem potencialmente substituir as células que são perdidas no processo de envelhecimento ou em processo de lesão e doença. A existência de células-tronco neurais (NSCs) foi primeiramente descrita por Reynolds e Weiss (1992) no adulto sistema central de mamíferos nervoso central (SNC), utilizando um novo sistema de cultura sem soro, o ensaio neurosphere (NSA). Com este ensaio, também é possível isolar e ampliar NSCs de diferentes regiões do SNC embrionárias. Estes, NSCs vitro expandidas são multipotentes e podem dar origem aos três principais tipos de células do SNC. Enquanto a NSA parece relativamente simples de executar, atenção para os procedimentos demonstrado aqui é necessário para alcançar resultados confiáveis ​​e consistentes. Este vídeo demonstra praticamente NSA para gerar e expandir NSCs partir do dia 14 de embriões de rato-tecido cerebral e fornece detalhes técnicos para que se possa atingir culturas neurosphere reprodutível. O procedimento inclui a colheita E14 embriões de camundongos, microdissecção cérebro para colher as eminências ganglionares, a dissociação do tecido colhido em meio NSC para ganhar uma suspensão de células individuais, e, finalmente, cultivo de células em cultura NSA. Depois de 5-7 dias em cultura, o que resulta neurospheres primários são várias passagens para expandir ainda mais o número do NSCs para futuros experimentos.

Watch this Scientific Journal Video about Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay at JoVE.com

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Este protocolo de vídeo demonstra a aplicação do ensaio neurosphere para o isolamento e expansão de células-tronco neurais do eminências ganglionares do dia 14 de embriões de rato-cérebro.

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

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

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Research

JoVE Journal

Neuroscience

24.5K visualizações.  Shiraz University of Medical Sciences, Shiraz, Iran. Este protocolo de vídeo demonstra a aplicação do ensaio neurosphere para o isolamento e expansão de células-tronco neurais do eminências ganglionares do dia 14 de embriões de rato-cérebro.

Vídeo: Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

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

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

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

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

Neurociência

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

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

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

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

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

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

Proteína localização em Cultura de Células-tronco neurais 3D: uma Metodologia de visualização híbrida

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neurais Colony-Forming Assay Cell: um ensaio para Discriminar Bona Fide células-tronco neurais de células progenitoras neurais

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Transplante de Células-Tronco cirúrgica do rato Neural na medula espinhal de camundongos infectados com o vírus da Hepatite neurotrófico rato

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

O Ensaio neuroblasto: um ensaio para a Geração e Enriquecimento de células progenitoras neuronais de Diferenciando Progeny Neural Stem Cell utilizando citometria de fluxo

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Enumeração de células-tronco neurais Usando clonais Ensaios

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolamento e Cultura de células estaminais adultas neurais do Subcallosal Zona Rato

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.