Este trabalho foi financiado pelo financiamento da Fundação Overstreet.

1. Itens que precisam ser preparados antes de prosseguir para Galvanização Cell: <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> Uma alíquota de NeuroCult NCFC meio isento de soro sem Citocinas é descongelado. </li><li> O meio é aquecido em banho-maria a 37 ° C. </li><li> Soluções estoque de fator de crescimento epidérmico (EGF), fator de crescimento básico de fibroblastos (b-FGF), na concentração de 10 mcg / mL e heparina em 0,2% estão dispostos à frente. </li><li> Dependendo do tamanho experimento, várias placas de cultura de 35 milímetros de tecido são necessários para a placa de células e um 150-200cm de plástico placa de Petri também é necessária para manter os pratos duplicar 35mm e um terceiro prato 35mm para água. </li></ol> 2. Preparação de células: Dependendo do seu experimento, as células podem ser preparados a partir de uma fonte adulta ou embrionária (tecido dissociado primária ou neurospheres dissociada). Dissecar os tecidos do adulto / embrionárias mouse do sistema nervoso central (SNC) ou dissociar neurospheres adulto / embrionárias derivadas de 1,2 como descrito anteriormente e, em seguida: <ol><li> A suspensão única célula é passado através de um filtro de malha 40 mícrons de tamanho, de modo a remover não-dissociada aglomerados. </li><li> 10μl da suspensão de células é misturado com 90μl de Trypan azul para realizar uma contagem de células. Nota: Outras diluições célula apropriada também pode ser usado. </li><li> Se estiver usando embrionárias primárias ou adultas derivadas de células neurais, diluir a suspensão de células a uma concentração de 6,5 x 10 5 células / mL em meio NSC completa. Se estiver usando células de neurospheres dissociada derivado de adulto ou embrionárias células neurais, diluir a suspensão de células a uma concentração de 2,2 x 10 5 células / mL em meio NSC completa. </li></ol> 3. Células chapeamento em meio semi-sólido N-CFCA: <ol><li> O volume apropriado dos seguintes componentes é misturado em ordem, dependendo do número de repetições. Aqui nós misturamos o montante necessário para duas repetições ou duplicar pratos. Para os números de repetições adicionais, consulte a tabela 1. <ul><li> 1,7 mL de NeuroCult NCFC meio isento de soro sem Citocinas </li><li> 330 mL de Suplementos NeuroCult NSC Proliferação </li><li> 6,6 mL de EGF (10μg/mL) </li><li> 32 mL de penicilina / estreptomicina </li><li> 3,3 mL de b-FGF (10μg/mL) necessária apenas se a cultura de células derivadas de células adultas neural. </li><li> 3,3 mL de heparina (0,2%) necessitaram de apenas se a cultura de células derivadas de células adultas neural. </li><li> 25μL da suspensão de células (de 2,2 x 10 5 células / mL de células neurospheres dissociada ou 6,5 x 10 5 células / mL pilhas de tecido do SNC dissociado). Nota: Os números célula final deve ser banhado a cerca de 2500 células por milímetro 35 placa de cultura para neurosphere células derivadas e 7500 células por 35 mm para placa de cultura de células primárias. Em alguns casos, a densidade de células de revestimento final, tem que ser ajustada através da realização de um experimento de titulação celular. Para as análises estatísticas, o número total de colónias detectadas após 21 dias de cultura deve estar dentro do intervalo de pelo menos 50-150 colônias. </li></ul></li><li> O meio contendo as células é misturado com cuidado e, em seguida, 1,3 mL de solução de colágeno frio é transferido para a suspensão de células e misturar bem pipetagem suave para evitar a introdução de bolhas. </li><li> A solução mista é dispensado para o centro de cada placa de cultura de 35 mm (~ 1,5 mL / prato) e os pratos estão inclinados suavemente com movimentos circulares para que a mistura espalhada uniformemente sobre a superfície do prato. . </li><li> A segunda via placas de cultura de 35 mm são colocados em uma placa de 100 milímetros Petri. A tampa do prato milímetros novo 35 é removido eo prato aberto também é colocado no prato de 100 milímetros mesma Petri. Água estéril é adicionado a este prato mm abrem 35 para manter a umidade ideal durante o período de incubação. Placas de bioensaio quadrado (245 mm) são usadas quando mais replicar 35 milímetros pratos foram criados. Mais uma vez, incluir 2 ou 3 abertas 35 pratos mm contendo água estéril. </li><li> As placas são transferidas para um conjunto de incubadora a 37 ° C, 5% CO2 e umidade de 95%. O colágeno congela por aumento da temperatura ea formação de gel irá ocorrer dentro de aproximadamente uma hora. As culturas não deve ser perturbado durante este tempo. </li><li> Células em cultura são incubados por 21 dias (diferenças de tamanho da colônia pode ser claramente distinguido após 21 dias). </li></ol> 4. Preparando Médio Replenishment e Alimentação da Cultura: Como N-CFCA culturas são incubados por um período de tempo prolongado (21 dias), as culturas devem ser alimentados com a NeuroCult apropriado completo médio de reposição preparado fresco da seguinte forma: <ul><li> 4,5 mL de NeuroCult NSC Basal Medium é misturado com 0,5 mL de Suplementos NeuroCult NSC Proliferação Nuclear e, em seguida, os fatores de crescimento é adicionado: </li><li> 250 L de 10μg/mL EGF (para dar uma concentração final de 0,5 mg / mL EGF) </li><li> 125 mL de 10μg/mL b-FGF (para dar uma concentração final de 0.25μg/mL b-FGF) necessária apenas se a cultura de células derivadas de células adultas neural. </li><li> 125 mL de heparina de 0,2%, necessária apenas se a cultura de células derivadas de células adultas neural. </li><li> 60 mL do Médio completo Replenishment NeuroCult apropriado (por células embrionárias ou adultas) é adicionado para o centro de cada prato Assay NCFC uma vez a cada sete dias durante a incubação cultura NCFC Assay inteiro (21 dias). </li></ul> 5. Colônias de pontuação N-CFC Assay Derivados e Cálculo da Freqüência de bona NSCs Fide e células progenitoras neurais: <ul><li> Cada placa de cultura 35 mm é colocado em um prato de pontuação em grade com o tamanho da grade de 2,0 mm x 2,0 mm e, em seguida, ambos os pratos são colocados no palco microscópio. </li><li> Usando baixa potência (2.5X - 5X) objetiva de cada prato é digitalizado e as colônias são pontuadas com base em seus tamanhos. </li><li> Colônias são classificadas em duas categorias principais: <ol><li> Menos de 2 mm de diâmetro </li><li> ≥ 2 mm de diâmetro </li></ol></li></ul> 6. Resultados representativos: Como no ensaio neurosphere, as células semeadas em N-CFC ensaio começam a proliferar e fazer pequenas colônias de células dentro de 3-7 dias após o revestimento (Figura 1). Dentro de duas semanas, as colônias de tamanhos diferentes podem ser distinguidos. Embora a maioria das colônias tende a parar de crescer após 14 dias, algumas colônias continuam a aumentar de tamanho. Ao dia 21, as colônias podem ser classificados em uma das quatro categorias: 1) menos de 0,5 mm de diâmetro, 2) 0,5-1 mm de diâmetro, 3) 1-2 mm de diâmetro e 4) ≥ 2mm de diâmetro. Praticamente, colônias menores que 2 mm de diâmetro são referidos como progenitor derivados (Figura 2) e colônias ≥ 2mm de diâmetro são denominados NSC derivados (Figura 3). O número de colónias ≥ 2mm de diâmetro por células totais chapeado, representa a freqüência da real bona fide células-tronco neurais de longo prazo auto-renovação e potencial multi-capacidades. O total de neurosphere formando freqüência em uma determinada população celular após 7-8 dias em um experimento paralelo NSA foi estimado para ser semelhante ao do total da colónia formando freqüência da população de células mesmo após 21 dias em um experimento de N-CFCA. O N-CFCA no entanto, oferece uma condição mais permissiva, de modo cada célula pode mostrar todo seu potencial proliferativo. <table border="1"><tbody><tr><td> Componente </td><td> 2 repetições </td><td> 3 repetições </td><td> 4 repetições </td></tr><tr><td> NeuroCult NCFC meio isento de soro sem Citocinas </td><td> 1700 </td><td> 2550 </td><td> 3400 </td></tr><tr><td> NeuroCult NSC Suplementos Proliferação </td><td> 330 </td><td> 495 </td><td> 660 </td></tr><tr><td> EGF (10 mcg / mL) </td><td> 6,6 </td><td> 9,9 </td><td> 13,2 </td></tr><tr><td> bFGF (10 mcg / mL), apenas para células adultas </td><td> 3,3 </td><td> 4,95 </td><td> 6,6 </td></tr><tr><td> Solução de heparina (0,2%), apenas para células adultas </td><td> 3,3 </td><td> 4,95 </td><td> 6,6 </td></tr><tr><td> Penicilina estreptomicina / (1:100) </td><td> 32 </td><td> 48 </td><td> 64 </td></tr><tr><td> Células em: 2,2 x 10 5 cultura de células / ml ou 6,5 x 10 5 células primárias / mL </td><td> 25 </td><td> 37,5 </td><td> 50 </td></tr><tr><td> Solução de colágeno </td><td> 1300 </td><td> 1950 </td><td> 2600 </td></tr></tbody></table> Tabela 1. Componentes da cultura ensaio completo N-CFC. <img src="/files/ftp_upload/2639/2639fig1.jpg" alt="Figura 1" /> Figura 1. Colônias Representante na N-CFCA cultura de NSCs uma passagem E14 rato sete dias após o plaqueamento. As colônias podem exibir morfologia diferente e tamanho. Ampliação original; 4x <img src="/files/ftp_upload/2639/2639fig2.jpg" alt="Figura 2" /> Figura 2. Colônias progenitor Representante derivados de diferentes tamanhos em N-CFCA cultura de uma passagem do mouse E14 NSCs 21 dias após o plaqueamento. Como mostrado, as colônias têm morfologia diferente, mas todos são mm menos 2 em tamanho. Ampliação original; 4x <img src="/files/ftp_upload/2639/2639fig3.jpg" alt="Figura 3" /> Figura 3. Representante bona fide de células-tronco derivadas colônia em N-CFCA cultura de uma passagem do mouse E14 NSCs 21 dias após o plaqueamento. Colônias de células-tronco derivadas podem exibir morfologia diferentes (vero vídeo), mas todos são ≥ 2mm de diâmetro. Ampliação original; 4x

<ol>
	<li>Azari, H., Rahaman, 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. , (2010).</li><li>Azari, H., Sharififar, S., Rahaman, M., Ansari, 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=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J Vis Exp. , (2011).</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, 1707-1710 (1992).</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. , (2008).</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>Louis, S. A., 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=Neurosphere+and+Neural+Colony-Forming+Cell+Assays.">Neurosphere and Neural Colony-Forming Cell Assays.</a> Protocols for Neural Cell Culture. 10, 1-28 (2010).</li></ol>

Um dos autores, Sharon A. Louis, é afiliado com StemCell Technologies Inc, produtora de alguns reagentes utilizados neste estudo.

Embora ensaio neurosphere 3,1,2 é o método mais comum para isolar e expandir as células-tronco neurais de uma variedade de fontes como adultas e embrionárias tecido do SNC, que não pode medir com precisão a freqüência NSC em uma população mista de células precursoras neurais (caule e progenitores) como não há uma relação de 12:59 entre o número de neurospheres eo número de bona fide células-tronco 4. Para resolver esta limitação, a NSA original foi adaptado de modo a permitir o tronco neurais e progenitores a crescer a sua capacidade de proliferação integral, durante três semanas 5-7. Ao contrário da NSA líquido, na N-CFCA as colônias são realmente clones derivados, como a matriz de colágeno semi-sólido impede a migração das células único banhado e agregação de colônias. Para obter resultados consistentes com este ensaio, recomendamos: <ol><li> Garantir uma suspensão única célula na suspensão celular original. Passe sua suspensão única célula através de um filtro de malha 40 mícrons de tamanho, de modo a remover não-dissociada aglomerados. </li><li> Manter a solução de colágeno no gelo ou no frigorífico 4 C °. O colágeno é o último item a ser adicionado à mistura de suspensão de células, uma vez que congela, aumentando a temperatura. </li><li> Não se esqueça de alimentar a cultura a cada semana. Adicionando o meio contendo reposição do fator de crescimento (s) uma vez a cada sete dias irá permitir que as células continuam a proliferar durante o período de cultura prolongada. </li><li> A densidade de células de revestimento final, foram otimizados para células derivadas de células primárias ou neural ou cultivadas de modo que o número total de colónias detectadas após 21 dias de cultura na NCFC-A está dentro da faixa de pelo menos 5-20 colônias. Esta gama permite a detecção dos raros NSC colônias derivadas&gt; 2mm de diâmetro em amostras ao fornecer um número suficiente de colônias para análises estatísticas. Dependendo da fonte de célula inicial, um número significativamente menor (&lt;50) e um maior número de colônias (&gt; 250) são por vezes obtidos. Colônias muito poucos resultará nas colônias&gt; 2mm de diâmetro sendo abaixo dos níveis de detecção, enquanto colônias demais resultará em anddepletion superlotação dos componentes do crescimento a médio, resultando em uma contagem de colônias imprecisas. Portanto, a densidade de células de revestimento deverá ser ajustada em conformidade (isto é, aumentando ou diminuindo o número total de células plated) </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NeuroCult NSC Basal Medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05700</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>STEMCELL</td>
<td>05701</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NCFC medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05720</td>
<td>&nbsp;</td>
<tr>
<td>0.05% 	trypsin-EDTA</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25300-062</td>
<td>&nbsp;</td>
<tr>
<td>Soybean trypsin inhibitor </td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6522</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer </td>
<td>Sieve</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>T25 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>136196</td>
<td>&nbsp;</td>
<tr>
<td>T80 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>178905</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>Reagent </td>
<td>STEMCELL</td>
<td>04902</td>
<td>&nbsp;</td>
<tr>
<td>EGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>2028-EG</td>
<td>&nbsp;</td>
<tr>
<td>b-FGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>3139-FB</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>Growth factor</td>
<td>Sigma</td>
<td>H4784</td>
<td>Reconstituted in PBS</td>
</tr>
<tr>
<td>15 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>35 mm culture dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27100</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27500</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

neural-colony forming cell assay: an assay to discriminate bona fide neural stem cells from neural progenitor cells

O ensaio neurosphere (NSA) é um dos métodos mais usados ​​para isolar, expandir e também calcular a freqüência de células-tronco neurais (NSCs). Além disso, este sistema de cultura sem soro também tem sido usado para expandir células-tronco e determinar sua freqüência a partir de uma variedade de tumores e tecidos normais. Foi demonstrado recentemente que um relacionamento um-para-um não existe entre a formação neurosphere e NSCs. Isto sugere que a NSA como actualmente aplicado, superestima a freqüência de NSCs em uma população mista de células precursoras neurais isoladas tanto o cérebro embrionárias e adultas dos mamíferos. Este vídeo demonstra praticamente um romance baseado colágeno semi-sólido ensaio, a neural-formadoras de colônia ensaio de células (N-ACCP), que tem a capacidade de discriminar-tronco a partir de células progenitoras com base em seu potencial de longo prazo proliferativa, e, portanto, fornece um método para enumerar freqüência NSC. No N-CFCA, colônias ≥ 2 mm de diâmetro são derivadas de células que cumprem todos os critérios funcionais de NSC, enquanto colônias &lt;2mm são derivadas de células progenitoras. O procedimento de N-CFCA pode ser usado para as células preparadas a partir de diferentes fontes, incluindo adultos primário e cultivadas ou células do SNC embrionárias de camundongos. Aqui usamos células preparadas a partir de uma passagem neurospheres gerados a partir de dia embrionário 14 cérebro de ratos para realizar N-CFCA. As culturas são reabastecidos com meio de proliferação a cada sete dias durante três semanas para permitir que as células banhado a exibir todo o seu potencial proliferativo e, em seguida, a freqüência de progenitoras neurais e de boa-fé células-tronco neurais são calculados, respectivamente, por contagem do número de colônias que são &lt;2mm e os que estão ≥ 2mm em referência ao número de células que foram inicialmente banhado.

Watch this Scientific Journal Video about Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells at JoVE.com

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

Este protocolo vídeo demonstra como discriminar e enumerar bona fide células-tronco neurais em uma população mista de células precursoras neurais utilizando o ensaio de célula neural formadoras de colônias.

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). ...

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Universidad San Sebastian

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JoVE Journal

Neuroscience

22.0K visualizações.  University of Florida. Este protocolo vídeo demonstra como discriminar e enumerar bona fide células-tronco neurais em uma população mista de células precursoras neurais utilizando o ensaio de célula neural formadoras de colônias.

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

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 ...

Neurociência

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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 span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

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 ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Em células vivas, imagens de células precursoras de órgãos sensoriais na Intact Drosophila As pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

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 ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Derivação sem alimentador de células da crista neural progenitoras pluripotentes humanas de células-tronco

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

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

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

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

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

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

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. ...

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.