Work was funded by a Development and Promotion of Science and Technology scholarship from the Thai Ministry for Education to W.W. and grants from Tenovus and the Anonymous Trust to M.P.S.

1. Preparação de mídia NOTA: O protocolo baseia-se na utilização de uma mistura de dois meios separados: DMEM / F12 suplementado com suplemento N2 modificado e Neurobasal suplementado com suplemento B27, tipicamente numa proporção de 1: 1. <ol><li> Prepara-se o suplemento N2 modificado por mistura dos componentes num tubo de 15 ml. Não vórtice ou filtrar este; misturar por inversão do tubo até a solução ficar clara. <ol><li> Iniciar pipetando 7,2 ml de DMEM / F12, em seguida adicionar 1 ml de 25 mg / ml de insulina (fez-se em 0,01 M de HCl) e misturar bem por inversão do tubo. É preciso um par de minutos até que a solução é clara. </li><li> Adicionar 1 ml de 100 mg / ml de apo-transferrina (preparados em água), 33 ul de 0,6 mg / ml progesternone (confeccionados a partir de etanol), 100 uL de 160 mg / ml (1 M) putrescina (preparados em água ), 10 ul de 3 mM de selenito de sódio (preparados em água) e 666 ul de 7,5% de albumina de soro bovino e misturar bem o tubo. Alíquota em 2,5 ml e armazenar a -20 °C por até 2 meses. </li></ol></li><li> Prepare a mídia. <ol><li> Para 250 ml de meio DMEM / F12 adicionar 2,5 ml de N2. Misture bem, mas não agite ou filtro. </li><li> Para 250 ml de Neurobasal meios adicionar 5 ml de suplemento de B27. Misture bem, mas não agite ou filtro. </li><li> Misture a mídia da passos 1.2.1 e 1.2.2 em uma proporção de 1: 1. Em alguns casos, uma mistura 1: mistura 3 pode ser necessárias (suplementado como o 1: 1 mistura no passo 1.2.4). </li><li> Para a mistura de adicionar 0,5 mL de L-glutamina (concentração final 0,2 mM) e 3,5 ul de 2-mercaptoetanol (concentração final 0,1 mM) e misturar bem, sem agitação. Armazene a mídia a 4 ° C por até 3 semanas e evitar a exposição à luz. Esta mistura final é chamada N2B27. </li></ol></li><li> Preparar a solução de gelatina. <ol><li> Prepara-se uma solução de gelatina a 1% em água ultrapura e autoclave a 121 ° C, 15 psi, durante 15 min. É importante que o frasco utilizado para este está completamente limpa de detergentes ou os desinfectantes, então recomenda-se que uma nova garrafa éutilizado para isso e é sempre apenas lavado em água ultrapura entre utilizações. Alíquota da solução a 1% em 50 ml de partes alíquotas e manter a 4 ° C, durante meses. </li><li> Aquece-se uma alíquota de 1% de gelatina em um banho de água a 37 C ° até se dissolver e adicionar a 500 ml de solução salina tamponada com fosfato quente (PBS). Misturar bem e pipetar suficiente para cobrir o fundo de cada poço ou placa. Permitir que a gelatina para revestir a superfície durante pelo menos 30 min. </li></ol></li></ol> 2. plaqueamento das células NOTA: Este protocolo se aplica igualmente a ESC rato cultivadas em 10% de soro com fator inibidor de leucemia (LIF), a substituição de soro com LIF ou meios livres de soro com proteína LIF e morfogenética óssea 4 (BMP4) ou inibidores de MEK e GSK3 (media 2i) com ou sem LIF. No entanto, o momento ea eficiência da diferenciação pode variar dependendo da mídia e células (ver discussão). Para os experimentos mostrados aqui usamos a linha 46C rato ESC (que tem um repórter EGFP batido om dos alelos Sox1 endógenos), cultivadas em GMEM com 10% de soro e LIF. Para melhores resultados, é importante que as células são dissociadas e replated na mídia N2B27; simplesmente mudando de mídia a partir de GMEM / soro / LIF para N2B27 sempre resulta em uma redução da eficiência de diferenciação em relação ao repique das células. <ol><li> Cresça as células em GMEM com 10% de soro, 1 mM de piruvato de sódio, aminoácidos não essenciais 1x, 0,1 M de 2-mercaptoetanol, e 100 unidades / ml de LIF 13. Para obter uma cultura subconfluentes de rato ESC, placa 1 x 10 6 células em uma cultura frasco T25, que terá 2-3 dias. </li><li> Observar células sob microscópio de campo brilhante. Quando as células são subconfluentes e pronto para passagem, lave-os duas vezes em PBS. Adicionar 1 ml de reagente de dissociação de células e retornar para o incubador durante 2-5 min. Embora tripsina e outras enzimas podem também ser usados ​​para dissociação de células, podem ter um impacto negativo sobre a ligação de células de modo que as densidades de plaqueamento terá de ser ajustÃsted. </li><li> Uma vez que as células começam a levantar a partir da placa, toque no vaso para desalojar-los em uma suspensão de células individuais. Recolher as células para um total de 10 ml de meio de dissociação de células e reagente e pipeta para um tubo de centrífuga de 15 ml. </li><li> Rodar as células a 300 xg durante 3 minutos à temperatura ambiente. </li><li> Aspirar cuidadosamente o sobrenadante e ressuspender o sedimento em 10 ml de N2B27 pré-aquecido por pipetagem para cima e para baixo 3-5 vezes. Quando pipetando a suspensão para baixo, da pipeta contra o lado do tubo para evitar a criação de bolhas. Contar as células com um contador de células automatizado ou um hemocitómetro e registar a concentração. </li><li> Prepara-se uma suspensão de células contendo o número desejado de células por unidade de volume a ser plaqueadas por cavidade em N2B27 pré-aquecido. Uma densidade de 10.500 células por cm 2 de um prato de 6 poços (isto é, 1 x 10 5 células por poço de um prato de 6 poços) é o ideal. Ver Tabela 1 para o número de células sugerido por cm 2 e PLAting volumes de suporte para uma variedade de recipientes de cultura de célula. </li><li> Aspirar a gelatina a partir do vaso de cultura e placa a suspensão de células de acordo com a densidade e volume de sugerido na Tabela 1. Não rodar as placas de que este vai concentrar-se as células para o centro. Coloque as culturas numa incubadora húmida a 37 ° C, 5% de CO 2. </li><li> Mudança de mídia a cada 1-2 dias, a substituição de todos os meios de comunicação com N2B27 fresco. Pipeta suavemente como a lavagem das células pode afetar a densidade e diminuir a eficiência de diferenciação nos dias seguintes. Onde viabilidade inicial após o plaqueamento é pobre, placa as células em uma 1: 3 mistura dos meios de comunicação N2 e B27-suplementados e mudar isso para N2B27 após os dois primeiros dias. As células começam a diferenciar e deve mostrar expressão Sox1 (visível por fluorescência verde do repórter na linha celular utilizada aqui 46C) dentro de 4-6 dias. </li></ol> 3. Immunofluorescent Coloração NÃOTE: O protocolo pode ser aplicado a qualquer tipo de navio. O volume de cada reagente é descrito por poço da placa de 6 poços e pode ser dimensionada para qualquer área de superfície. Repique não é recomendada devido à viabilidade baixa depois. <ol><li> Remover meio de diferenciação e fixar as células por adição de 500 ul de 4% (v / v) de paraformaldeído, deixar durante 15 min. </li><li> Lavar e permeabilizar as células com 2 ml de PBS-T (solução salina tamponada com fosfato com 0,5% (v / v) de Tween-20) por duas vezes. As células podem ser mantidas em PBS-T, durante até 2 meses a 4 ° C. </li><li> Substituir PBS-T com 1 ml de PBS-T com 10% (v / v) de soro (a partir da mesma espécie que o anticorpo secundário). Incubar 1 hora sobre o roqueiro placa à temperatura ambiente para bloquear qualquer ligação não específica. </li><li> Após 1 h, lavar as células duas vezes com 2 ml de PBS-T, e incuba-se 500 ul de IgG de ratinho contra βIII-tubulina (diluída 1: 1000 em PBS-T, com 10% de soro). Colocar no prato oscilante, e deixar O / N a 4 ° C. </li><li> A partir deste passo, sempre proteger o vasoda luz. Lavar as células duas vezes com PBS-T, em seguida, adicionar 500 ul de anticorpo anti-IgG de ratinho marcado com fluorescência (diluída a 2 ug / ml em PBS-T, com 10% de soro). Colocá-lo sobre a placa oscilante durante 1 h à TA. </li><li> Lavar as células duas vezes com PBS-T, em seguida, adicionar 500 mL de DAPI (diluído para 0,5 ug / ml em PBS-T) e deixar durante 5 min. </li><li> Lavam-se as células novamente e mantê-las em PBS-T. As células estão prontas para ser fotografado e pode ser mantido por até 2 semanas a 4 ° C. Note que o sinal GFP desaparece ao longo do tempo, por isso é inadequado para comparar a intensidade GFP de células fixas e ao vivo ou de células fixadas em momentos diferentes. </li></ol>

<ol>
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Biol. 168, 342-357 (1995).</li><li>Fraichard, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro.+differentiation+of+embryonic+stem+cells+into+glial+cells+and+functional+neurons.">In vitro. differentiation of embryonic stem cells into glial cells and functional neurons.</a> J. Cell Sci. 108, 3181-3188 (1995).</li><li>Strubing, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+pluripotent+embryonic+stem+cells+into+the+neuronal+lineage+in+vitro+gives+rise+to+mature+inhibitory+and+excitatory+neurons.">Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons.</a> Mech. Dev. 53, 275-287 (1995).</li><li>Stavridis, M. P., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+mouse+embryonic+stem+cells.">Neural differentiation of mouse embryonic stem cells.</a> Biochem Soc Trans. 31, 45-49 (2003).</li><li>Ying, Q. L., Stavridis, M., Griffiths, D., Li, M., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+embryonic+stem+cells+into+neuroectodermal+precursors+in+adherent+monoculture.">Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.</a> Nat Biotechnol. 21, 183-186 (2003).</li><li>Ying, Q. L., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+conditions+for+neural+commitment+and+differentiation.">Defined conditions for neural commitment and differentiation.</a> Methods Enzymol. 365, 327-341 (2003).</li><li>Stavridis, M. P., Lunn, J. S., Collins, B. J., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+period+of+FGF-induced+Erk1/2+signalling+is+required+for+vertebrate+neural+specification.">A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification.</a> Development. 134, 2889-2894 (2007).</li><li>Stavridis, M. P., Collins, B. J., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+orchestrates+fibroblast+growth+factor+signalling+to+drive+embryonic+stem+cell+differentiation.">Retinoic acid orchestrates fibroblast growth factor signalling to drive embryonic stem cell differentiation.</a> Development. 137, 881-890 (2010).</li><li>Speakman, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+O-GlcNAc+Levels+Activate+Epigenetically+Repressed+Genes+and+Delay+Mouse+ESC+Differentiation+Without+Affecting+Naive+to+Primed+Cell+Transition.">Elevated O-GlcNAc Levels Activate Epigenetically Repressed Genes and Delay Mouse ESC Differentiation Without Affecting Naive to Primed Cell Transition.</a> Stem Cells. 32, 2605-2615 (2014).</li><li>Kunath, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+stimulation+of+the+Erk1/2+signalling+cascade+triggers+transition+of+pluripotent+embryonic+stem+cells+from+self-renewal+to+lineage+commitment.">FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.</a> Development. 134, 2895-2902 (2007).</li><li>Dani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+induction+of+stem+cell+renewal+by+LIF-deficient+cells:+a+new+ES+cell+regulatory+pathway.">Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway.</a> Dev Biol. 203, 149-162 (1998).</li><li>Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+differentiation+of+embryonic+stem+cells.">Culture and differentiation of embryonic stem cells.</a> Journal of Tissue Culture Methods. 13, 89-94 (1991).</li><li>Aubert, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+mammalian+neural+genes+via+fluorescence-activated+cell+sorter+purification+of+neural+precursors+from+Sox1-gfp+knock-in+mice.">Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 100, 11836-11841 (2003).</li><li>Niwa, H., Burdon, T., Chambers, I., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+of+pluripotent+embryonic+stem+cells+is+mediated+via+activation+of+STAT3.">Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.</a> Genes Dev. 12, 2048-2060 (1998).</li><li>Silva, J., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+pluripotency.">Capturing pluripotency.</a> Cell. 132, 532-536 (2008).</li></ol>

The authors have nothing to disclose.

O protocolo de diferenciação neural monocamada tem sido usado por mais de uma década 6. O protocolo é altamente eficiente, composto de meio definido, e feito de um sistema de monocamada que torna o sistema mais aplicável para pré-clínica (por exemplo, o rastreio de fármacos) usa. No entanto, existem alguns factores críticos que determinam a eficiência de diferenciação. Este artigo aponta esses fatores ea solução para cada obstáculo. Densidade das células a...

As células estaminais embrionárias são células pluripotentes derivadas do embrião com a capacidade de proliferar indefinidamente in vitro, mantendo a capacidade de se diferenciar em todos os tipos de células dos adultos após reintrodução num embrião fase adequada (através da formação de uma quimera), injecção em hospedeiros singénicos ou imunocomprometidos (através da formação de um teratoma) ou in vitro sujeitos a estímulos apropriados 1. A diferenciação in vitro de células estaminais embrionárias de ratinho em linhagens neurais foi descrita pela primeira vez em 1995 e envolveu a formação de agregados multicelulares suspensão (corpos embrióides, EBS) em meio contendo soro suplementado com o ácido retinóico morfogénio 2-4. Uma vez que, em seguida, uma variedade de protocolos têm sido desenvolvidos para permitir a diferenciação neuronal 5. Muitos ainda utilizar agregação, os outros co-cultura com indução de tipos de células e vários envolvem a utilização de meios isentos de soro. Todos os protocolos tem vantagens umand desvantagens e a natureza precisa da neural ou células neuronais produzida também varia de acordo com o protocolo utilizado. O protocolo ideal seria robusta, escalável e fazer uso de meios totalmente definidos e substratos, ser passíveis de monitorização não invasiva do processo de diferenciação e resultar na geração de populações puras de células progenitoras neurais capazes de ser modelado por sinais externos e para diferenciar em todos os subtipos neuronais e gliais com elevada eficiência e rendimento em relativamente pouco tempo. Nos últimos doze anos, temos vindo a utilizar um método para gerar progenitores neurais e neurônios de rato ESC em uma baixa densidade, cultura monocamada aderente sem soro 6-10. Este protocolo cumpre muitos dos critérios enunciados anteriormente: em nossas mãos a eficiência de diferenciação tem sido bastante consistente ao longo de muitos anos e uma variedade de linhas de células, ele pode ser escalado para cima ou para baixo (que usamos com sucesso navios de placas de 96 poços para 15 cm de diâmetro diela é) e os meios usados ​​são bem definidos. O processo é passível de microscopia Timelapse para o controlo da diferenciação e uma variedade de padrões de sinais pode ser adicionado para induzir a geração de tipos distintos de subtipos neuronais (por exemplo, a Shh e Fgf8 para neurónios dopaminérgicos 6). No entanto, existem alguns desafios para o sucesso da aplicação deste protocolo. Um dos aspectos fundamentais é uma preparação cuidadosa da mídia. Nós sempre preparar a mídia in-house, apesar da disponibilidade de alternativas comerciais. Um dos suplementos usados ​​(N2; ver Protocol) tem modificações sobre o padrão versões disponíveis no mercado. Finalmente, uma das etapas mais importantes para a aplicação bem sucedida deste método é a densidade celular óptima para o chapeamento. Isto é principalmente devido ao passo que a natureza autócrina de um dos sinais indutores (FGF4 11) requer que as células estão presentes suficientes para permitir viabil óptimadade e diferenciação, em muito altas densidades diferenciação é prejudicada (possivelmente em parte devido à produção autócrina de LIF 12). Portanto, é importante que a preparação ambos os meios e revestimento celular são realizadas com cuidado e de forma consistente para assegurar os melhores resultados.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, serum free</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>Reconstitute with sterile 0.01M HCl</td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>Reconstitute with ethanol</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V</td>
			<td>Life Technologies</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td>Make sure it is completely dissolved before use as glutamine is usually sedimented</td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>GMEM</td>
			<td>Life Technologies</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukaemia inhibitory factor</td>
			<td colspan="3">prepared in-house as in Smith 1991 Journal of Tissue Culture Methods</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Mouse anti betaIII tubulin IgG antibody</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence-labelled anti-Mouse IgG antibody</td>
			<td>Life Technologies</td>
			<td>A31571</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>25cm2 tissue culture flask</td>
			<td>Thermo Scientific</td>
			<td>156367</td>
			<td></td>
		</tr>
	</tbody>
</table>

neural differentiation of mouse embryonic stem cells in serum-free monolayer culture

A capacidade de diferenciação de células estaminais embrionárias de ratinho (ESC) para progenitores neurais permite o estudo dos mecanismos que controlam especificação neural, bem como a geração de tipos de células neuronais maduros para estudo posterior. Neste protocolo, descrevem um método para a diferenciação de ESC para progenitores neurais utilizando, cultura em monocamada sem soro. O método é escalável, eficiente e resulta num rendimento de ~ 70% de células progenitoras neurais dentro de 4-6 dias. Ele pode ser aplicado a partir de várias estirpes CES cultivadas sob uma variedade de condições. Progenitores neurais podem ser autorizados a diferenciar ainda mais em neurônios funcionais e glia ou analisados ​​por microscopia, técnicas de citometria de fluxo ou moleculares. O processo de diferenciação é passível de microscopia de lapso de tempo e pode ser combinada com o uso de linhas de repórter para monitorizar o processo de especificação neural. Nós fornecemos instruções detalhadas sobre a preparação de meios e optimização densidade celular para permitir que o processo abe aplicado a maioria das linhas de ESC e uma variedade de recipientes de cultura de célula.

Nesta experiência, utilizou-se a linha de células de 14 46C, células estaminais embrionárias de ratinho com um endógeno Sox1-repórter GFP, para controlar a diferenciação neuronal. Utilizando esta linha celular, expressão de Sox1, um marcador para células progenitoras neurais, pode ser detectado por fluorescência verde. Chapeamento de densidade é um fator crítico para alcançar a diferenciação neuronal. Células estaminais embrionárias de ratinho foram plaqueadas em placas de 6 poços em difere...

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Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Diferenciação Neural of Mouse Células-Tronco Embrionárias em soro livre de cultura em monocamada

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

neural-differentiation-mouse-embryonic-stem-cells-serum-free

Research

JoVE Journal

Developmental Biology

14.0K visualizações.  University of Dundee. This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Vídeo: Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

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

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

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

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

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

Biologia do Desenvolvimento

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Regulação epigenética da Cardiac diferenciação de células estaminais embrionárias e Tecidos

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Um Modelo de enzimas e Cultura de Células-Tronco Neural Serum-livre para EMT Investigation Adequado para Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Produção em larga escala de cardiomiócitos a partir de células-tronco pluripotentes humanas utilizando um altamente reprodutível pequeno-Based Molecule Protocolo de Diferenciação

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Dirigido a diferenciação de progenitores hematopoiéticos primitivo e definitiva de células pluripotentes humanas

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Análise do induzida por ácido retinóico Neural diferenciação de células estaminais embrionárias rato em corpos embrióides Dois e tridimensionais

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Rápida, dirigida a diferenciação de células epiteliais de pigmento Retinal de células-tronco humanas embrionárias ou induzida de Pluripotent

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Geração de células-tronco neurais induzidas de células mononucleares periféricas e diferenciação para precursores de neurónios dopaminérgicos para estudos de transplante

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

Produção livre de soro de Hepatesferas humanas tridimensionais de células-tronco pluripotentes

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Diferenciação neural eficiente usando a cultura unicelular de células-tronco embrionárias humanas

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...