Este manuscrito é dedicado à memória do Dr. Wenlan Wang, que faleceu em 26 de maio de 2011. Agradecemos ao Dr. Douglas A. Kerr para generosamente fornecer os HBG3 células MES utilizados neste estudo. Este trabalho foi financiado por Nemours, uma bolsa (2 RR016472-10) no âmbito do programa INBRE do Centro Nacional de Pesquisa de Recursos (NCRR), e um COBRE Prêmio da NCRR (5 P20 RR020173-05) para apoiar o Centro de Pesquisa Pediátrica do I. Alfred duPont Hospital for Children, Wilmington, Delaware, EUA.

1. Passo 1: Mouse Embryonic Stem Cultura de Células (MES) (Timing: 3 dias) 1. Plating primárias embrionárias fibroblastos de rato (PMEF) <ol><li> Revestimento quatro 100-mm de tecido pratos de cultura com a gelatina, para adesão PMEF. Adicionar 8 ml de 0,1% de solução de gelatina (StemCell Technologies) a cada prato e incubar durante 30 min à temperatura ambiente. </li><li> Retirar o excesso de gelatina a partir de pratos. Não lave os pratos. </li><li> Diluir um frasco de Mitomicina C-inactivado PMEF (Millipore) que contém aprox. 5 x 10 6 células em 40 ml de PMEF meios (ver receita na Tabela 1) ea placa para quatro 100-mm pratos de cultura de tecidos. O PMEF proporcionar uma camada alimentadora de células MES. </li><li> PMEF deve ser revestida pelo menos um dia antes da adição de culturas de células de MES. PMEF pode ser usada até 1 semana após o plaqueamento. </li></ol> 2. Cultivo de células MES Para monitorizar a eficiência do MES diferenciação celular em neurónios motores, Utilizou-se HBG3, uma linha de células ES derivado de um ratinho transgénico expressando eGFP sob o controlo do promotor de neurónio motor específico Hb9. <ol><li> Degelo 1 frasco de HBG3 células MES (aprox. 1 x 10 7, pelo Dr. Douglas Kerr, da Universidade Johns Hopkins), em banho-maria a 37 ° C. Adicionar 5 ml de meios ES (ver receita na Tabela 1) em um tubo de 15 ml e girar para baixo células a 800 rpm durante 5 min. </li><li> Aspirar o sobrenadante e Ressuspender as células MES em 20 ml de meio recém-acrescentado ES com factor de Leucemia inibitória (LIF), a uma concentração final de 10 ng / ml. NOTA: LIF deve ser adicionado no dia da utilização e é necessária para manter as células MES em um estado indiferenciado. </li><li> Remover 10 ml de meio PMEF a partir do PMEF em cada prato e adicionar 10 ml da suspensão de células ES HBG3 para cada prato. </li><li> 24 h após o plaqueamento, as células de MES formar colónias pequenas, semelhantes em tamanho para a colónia indicado pela seta na Figura 1B. 72 h após o plaqueamento, Coloniaumento es em tamanho, muitos sendo de aproximadamente 100 um de diâmetro (Figura 1B, indicado pelas setas). Estas células estão prontas para a diferenciação. Os meios devem ser substituídos por dia para manter a proliferação de células. </li></ol> 2. Passo 2: Indução Neural (Timing: 2 dias) Para induzir a diferenciação de células de MES em neurónios motores, as células de MES precisam de ser separado do PMEF e cultivado em um ambiente de suspensão. Gelatina frascos revestidos são utilizados para separar PMEF a partir de células de MES. <ol><li> Quando as colónias MES estão prontos para a indução neural (definido como o dia diferenciação 0), o revestimento 2 T150 frascos com 0,1% de gelatina (18 ml / balão) durante 30 min à temperatura ambiente e depois remover o excesso de gelatina e lavar com PBS 1X três vezes. NOTA: Cada cultura prato de 100 mm vai precisar de um balão T150-gelatina revestido para separar PMEF. </li><li> Cuidadosamente remova a mídia por aspiração, tomando cuidado para não desalojar colônias frouxamente conectados. Adicionar 10 ml breve PBSly e remova por aspiração para concluir a etapa de lavagem. </li><li> Para separar as células de MES a partir de PMEF, adicionar 0,25% de tripsina / EDTA (3 ml / prato, StemCell Technologies) e incubar durante 3-5 min a 37 ° C até que as células-tronco e PMEF destacar a partir da placa. Adicionar 15 ml de meio ES frescos sem LIF e células de pipeta para cima e para baixo para quebrar colônias (cerca de 15-20 vezes). Em seguida, adicionar a uma 0,1% de gelatina revestido T150 balão e incubar durante 30 min a 37 ° C. Após a incubação, PMEF deve anexar à superfície gelatinizada, enquanto as células-tronco embrionárias devem estar flutuando. Recolher as células flutuantes MES para um tubo de 50 ml. </li><li> Girar para baixo a 800 rpm e Ressuspender as células em 10 ml de Meio de Diferenciação Neural (ver receita na Tabela 1). </li><li> Contar as células utilizando um hemocitómetro e, em seguida, placa aproximadamente 2 x 10 6 células MES para um prato de cultura de 100 mm bacteriana ou suspensão. Estas placas de permitir o crescimento de culturas em suspensão e promover a formação de corpos embrióides (EBS). </li><li> Em diferenciaçãoção dia 1, as células de MES formar pequenos agregados flutuantes (EBS) que são visíveis sob um microscópio de luz. Qualquer PMEF transição anexar ao prato. Transferir as células em suspensão e meios de comunicação para um tubo de 15 ml e centrifugar a baixa velocidade (500 rpm) durante 3 min. Aspirar meios cuidadosamente, adicionar 10 ml de Meio fresco Diferenciação Neural ao EBS peletizadas e, em seguida células da placa em um prato de novo bacteriana. Além disso para reabastecer os meios de comunicação, este passo remove qualquer PMEF que carregam a partir do passo anterior. </li><li> No dia 2 diferenciação, EBs está pronto para ser induzido em neurónios motores. </li></ol> 3. Passo 3: Motor Neuron Especificação (Cronograma: 5 dias) <ol><li> No dia 2 de diferenciação, redemoinho da placa de cultura e transferir os meios de comunicação e EBS em um tubo de 15 ml. Deixe EBs resolver por gravidade (~ 10 min) ou por centrifugação a baixa velocidade (500 rpm) durante 3 min. </li><li> Aspirar cuidadosamente médio e ressuspender EBs em 10 ml de Motor Neuron Diferenciação (MND), média (ver receita em <strong&gt; Tabela 1). Placa EBs em um prato de novo bacteriana e alterar os meios de comunicação por dia durante 5 dias. </li><li> No dia 7 diferenciação, EBS deve ser de aproximadamente 150 a 200 um de diâmetro e deve expressar sinais fortes GFP (Figura 1C). Estes EBs está pronto para ser dissociada e banhado em poly-DL-ornithine/laminin pratos revestidos ou lamelas. </li></ol> 4. Etapa 4: Preparação de Poly-DL-ornithine/laminin Lamelas revestidos (Timing: 2 dias) Dois dias antes dissociar EBs (ou seja, no dia diferenciação 5), preparar as lamínulas poly-DL-ornithine/laminin-coated. <ol><li> Lamelas lugar 12-mm de diâmetro (Warner Instruments) no fundo de uma placa de 24 cavidades. Dissolve-se 25 mg de poli-DL-ornitina (Sigma-Aldrich) em 25 ml estéril, água bidestilada (d 2 H2O) para se obter uma solução-mãe 10X (1 mg / ml). Quando pronto para usar, fazer uma diluição 1/10 de poli-DL-ornitina com d 2 H2O para se obter concentração de 100 ug / ml. Adicionar 0,5 ml a cada poço da placa de 24 poços e incubar a 4 ° C durante a noite. </li><li> No dia seguinte (dia diferenciação 6), aspirado poli-DL-ornitina e deixar as placas e de ar de tampa desliza seco em uma capa de cultura de tecidos. Lavar os poços da placa com d 2 H2O três vezes. Deixar o ar seco durante mais uma hora. </li><li> Dissolve-se 1 mg rato laminina (Millipore) em 5 ml de gelado a frio 1X PBS para fazer a solução de estoque de 100x (200 ug / ml). Quando pronto para usar, fazer 1/100 de diluição em gelada-fria PBS 1X (2 ug laminina / ml). Adicionar 0,5 ml / poço numa placa de 24 poços e incubar em 4 ° C durante a noite. Antes de células de semeadura, laminina excesso deve ser removido e os poços lavados com PBS 1X duas vezes. Lamelas podem ser armazenados em MND médio (0,5 ml / poço). </li></ol> 5. Passo 5: Alongamento Axon (Timing: 2 dias) <ol><li> No dia 7 diferenciação, recolher EBs para um tubo de 15 ml e permitir EBS para resolver (aproximadamente 3 min). Aspirado meios de comunicação e re-suspend EBs em PBS. Permitir EBs para resolver e, em seguida, aspirar PBS. Repetir este passo 3 vezes. Adicionar 1 ml de Accumax (Millipore) para o EBS, suavemente misturar e incubar durante 5 min à temperatura ambiente. Em seguida, aspirar Accumax excesso cobrindo o EBS. </li><li> Para dissociar EBs, adicionar 3 ml de meio de MND. Pipeta cima e para baixo 20 vezes usando uma micropipeta Eppendorf 1 ml (azul ponta) para romper EBs. Incubar durante 2 minutos à temperatura ambiente. Transferir suspensão de células para um tubo de milímetro 12x75 com célula de coador tampa (BD Falcon) para se obter uma suspensão de células individuais. Repetir este passo de dissociação com tufos restantes e células retidas pelo filtro, de modo a enriquecer o rendimento de células individuais. A suspensão celular final única será 6 ml. </li><li> Misturar cuidadosamente esta suspensão de células individuais e contar as células utilizando um hemocitómetro. Diluir as células em meio de MND (suplementado com 10 ng / ml de cada BDNF, GDNF, CNTF, e NT-3) a uma densidade de 2x10 células por 5 ml. Adicionar a suspensão de células (0,5 ml / poço) para placas de 24 poços contendo polamelas ly-DL-ornithine/laminin revestidos (previamente preparado como descrito acima). As células diferenciadas estender neurites longas após 2 dias em cultura (Figura 1D). </li></ol> 6. Os resultados representativos A Figura 1A mostra um esboço do protocolo. No passo 1, as células são cultivadas em MES PMEF e suplementado com LIF para impedir a diferenciação espontânea. Em geral, as colônias indiferenciadas MES são redondos e compactos, e definiram claramente bordas. As colónias não estão em contacto um com o outro. Tipicamente, as células de MES deve ser dividida em uma razão de 1:4 a cada 2 ~ 3 dias. No entanto, cada linha de células indivíduo vai crescer a uma taxa diferente e da razão de separação devem ser determinadas empiricamente. As setas na Figura 1B indicar a aparência típica de células MES após a cultura em uma camada de alimentador PMEF durante 3 dias. Estas colônias são grandes (50-100 m de diâmetro), mas ainda manter a ponta redonda e definidoss. Nesta fase as colónias estão prontos para a diferenciação ou subcultura. Uma ponta da flecha na Figura 1B mostra uma colônia MES pequena que você costuma encontrar 24 h após o plaqueamento. Quatro dias após o plaqueamento, as células tornam-se MES overgrown. Eles mostram uma aparência achatada e perda dos limites definidos, indicando que eles estão começando a diferenciar. Essas células não são ideais para diferenciação em células neuronais. Para diferenciar células MES em neurónios motores, as células de MES precisam ser cultivados em condições de suspensão para permitir a formação EB. No passo 2, as células ES são transferidos para uma superfície de cultura sem uma camada alimentadora para promover a formação de EB. Neste passo, são incubadas em meio suplementado com Diferenciação Neural Noggin, bFGF, e FGF-8 durante 2 dias para as células directos para uma linhagem neuronal. Pequeno EBS pode ser observado ao microscópio no dia 1 de diferenciação. Eles devem estar flutuando no meio. Esta transição PMEF também pode ser encontrada em dia 1de diferenciação e deve ser removido. Estas células aderir aos pratos bacterianas modo são eliminados quando EBS são passadas para um prato de novo bacteriana. Assim, por dia 2 de diferenciação, PMEF poucos ou não deve ser vista no prato de cultura. No passo 3, continuou transferência de EBS para novos pratos deve assegurar a remoção de qualquer PMEF restante. EBS são cultivadas em Motor Neuron de Diferenciação (MND) meios suplementados com AR e SAG durante 5 dias para diferenciar as células em neurónios motores. Durante a cultura, as EBs continuar a crescer em tamanho e pode ser visto a olho nu no dia diferenciação 3 ~ 4. Figura 1C mostra a aparência microscópica de EBS em dia diferenciação 7. Ao contrário das células indiferenciadas, EBs mostram forte fluorescência devido à expressão da GFP. Estes EBs são perfeitamente diferenciados e estão prontos para a dissociação. A citometria de fluxo demonstrou que 51% ± 0,8% das células a partir da EBs dissociado tinha diferenciadas em células que expressam GFP 5. No passo 5, a cultura de thneurónios ese motor na presença de GDNF, CNTF, BDNF, NT3 e resulta em extensão de longos projecções axonal. A Figura 1D mostra o aparecimento de células diferenciadas 2 dias após o plaqueamento da EBS dissociados. Observação neurites longas que se estendem a partir dos corpos celulares das células chapeados. Coloração imunofluorescente mostra que a GFP + de células expressam o marcador pan-neuronal (neurofilamento de cadeia média), e dois marcadores neuronais específicas a motor, (Islet-1 e de colina-acetiltransferase, a Figura 2). <img alt="A Figura 1" src="/files/ftp_upload/3813/3813fig1.jpg" /> Figura 1. Diferenciação das células MES em neurónios motores (figura modificado a partir do Wu et al. 5). A) Esquema do protocolo de diferenciação das células MES para os neurônios motores. B) As células indiferenciadas MES formar colónias redondas sobre o topo de uma camada alimentadora de fibroblastos. Eles têm fraca expressão da GFP. C) As células foram separadas do MES camadas alimentadoras e blovermelha em baixo de fixação pratos para formar EBs. Durante os primeiros dois dias do processo de diferenciação, as células foram expostas a 50 ng / ml Noggin, 20 ng / ml de bFGF, e 20 ng / ml FGF-8. Posteriormente, foram induzidas a diferenciar com um ácido retinóico iM e 1 uM sónica SAG agonista hedgehog durante 5 dias. As células diferenciadas MES expressa de fluorescência da GFP forte. D) Depois de 7 dias de diferenciação, as EBs foram dissociados e plaqueados em placas revestidas poly-DL-ornithine/laminin. As células diferenciadas alargado longos processos neuronais após 2 dias em cultura. Barra de escala = 200 mM. MN = neurônio motor. B, C e D são imagens de campo claro e B &#39;, C&#39; e D &#39;são correspondentes imagens de fluorescência. <img alt="A Figura 2" src="/files/ftp_upload/3813/3813fig2.jpg" /> Figura 2. Caracterização de neurónios MES células derivadas do motor. Técnica de imunofluorescência para a cadeia de neurofilamentos médio (NF-M, vermelho), Chat (vermelho), e Ilhota-1 (vermelho) foi coincidente com GFP (verde) expression. DAPI (azul) foi usado para identificar os núcleos. Barra de escala = 50 mM.

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A qualidade de células MES é o parâmetro mais crítico para a geração eficiente de neurónios motores. células de MES deve ser cultivado em PMEF para impedir a diferenciação espontânea. A adição de LIF ajuda a manter as células MES em um estado indiferenciado. Como as células de MES dividir cada h 12-15, o meio de cultura se torna empobrecido rapidamente e deve ser substituído por dia. Activação eficiente da via Shh é um parâmetro crítico necessário para induzir a motor e...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Concentração final </td></tr><tr><td> PMEF </td><td> Millipore </td><td> PMEF-H </td><td> NA </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> PMEF médio </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965-118 </td><td> NA </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 16140-071 </td><td> 10% </td></tr><tr><td> L-glutamina </td><td> Invitrogen </td><td> 25030-081 </td><td> 1% </td></tr><tr><td> A penicilina / estreptomicina </td><td> Invitrogen </td><td> 15240-063 </td><td> 1% </td></tr><tr><td></td><td></td&gt; <td></td><td></td></tr><tr><td> MES médio </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell Technologies </td><td> 36250 </td><td> NA </td></tr><tr><td> ES Cell-Qualificado FBS </td><td> Invitrogen </td><td> 16141-079 </td><td> 15% </td></tr><tr><td> Glutamax-I </td><td> Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> Non-aminoácidos essenciais </td><td> Invitrogen </td><td> 11140-050 </td><td> 1% </td></tr><tr><td> Nucleosídeos </td><td> Millipore </td><td> ES-008-D </td><td> 1% </td></tr><tr><td> β-mercaptoetanol </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> A penicilina / estreptomicina </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> LIF </td><td> Millipore </td><td&gt; LIF2010 </td><td> 10 ng / ml </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Meio de diferenciação neural </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell Technologies </td><td> 36250 </td><td> NA </td></tr><tr><td> ES Cult FBS </td><td> StemCell Technologies </td><td> 06905 </td><td> 15% </td></tr><tr><td> Non-aminoácidos essenciais </td><td> Invitrogen </td><td> 11140-050 </td><td> 1% </td></tr><tr><td> Mono-tio glicerol </td><td> Sigma-Aldrich </td><td> M-6145 </td><td> 1mM </td></tr><tr><td> Caneca pequena </td><td> Invitrogen </td><td> PHC1506 </td><td> 50 ng / ml </td></tr><tr><td> FGF-8 </td><td> Invitrogen </td><td> PHG0274 </td><td> 20 ng / ml </td></tr><tr><td> bFGF </td><td> Invitrogen </td><td> PHG0024 </td><td> 20 ng / ml </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> MND médio (diferenciação) </td><td></td><td></td><td></td></tr><tr><td> ES Cult-Médio-A Basal </td><td> StemCell Technologies </td><td> 5801 </td><td> NA </td></tr><tr><td> Substituição Knockout soro </td><td> Invitrogen </td><td> 10828-028 </td><td> 10% </td></tr><tr><td> N-2 suplemento </td><td> Invitrogen </td><td> 17502-048 </td><td> 1% </td></tr><tr><td> Seu suplemento B- </td><td> StemCell Technologies </td><td> 07155 </td><td> 1% </td></tr><tr><td> Ácido ascórbico </td><td> StemCell Technologies </td><td> 07157 </td><td> 1% </td></tr><tr><td> A penicilina / estreptomicina </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> Glutamax-I </td><td>Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> D-glucose </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% em d 2 H2O </td></tr><tr><td> A fibronectina </td><td> StemCell Technologies </td><td> 07159 </td><td> 5 ug / ml </td></tr><tr><td> Heparina </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 ug / ml em d 2 H2O </td></tr><tr><td> β-mercaptoetanol </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> Ácido Retinóico </td><td> Sigma-Aldrich </td><td> R-2625 </td><td> 1 uM </td></tr><tr><td> SAG </td><td> Merck Química </td><td> 566660 </td><td> 1 uM </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> MND médio (Motor Neuron cultura) </td><td></td><td></td><td></td></tr><tr><td> ES Cult-Médio-A Basal </td><td> StemCell Technologies </td><td> 5801 </td><td> NA </td></tr><tr><td> Substituição Knockout soro </td><td> Invitrogen </td><td> 10828-028 </td><td> 10% </td></tr><tr><td> N-2 suplemento </td><td> Invitrogen </td><td> 17502-048 </td><td> 1% </td></tr><tr><td> Seu suplemento B- </td><td> StemCell Technologies </td><td> 07155 </td><td> 1% </td></tr><tr><td> Ácido ascórbico </td><td> StemCell Technologies </td><td> 07157 </td><td> 1% </td></tr><tr><td> A penicilina / estreptomicina </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> Glutamax-I </td><td> Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> D-glucose </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% em d 2 H2O </td></tr><tr><td> A fibronectina </td><td> StemCTecnologias ell </td><td> 07159 </td><td> 5 ug / ml </td></tr><tr><td> Heparina </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 ug / ml em d 2 H2O </td></tr><tr><td> β-mercaptoetanol </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> BDNF </td><td> R &amp; D Systems </td><td> 248-BD-005/CF </td><td> 10 ng / ml </td></tr><tr><td> CNTF </td><td> R &amp; D Systems </td><td> 257-NY-010/CF </td><td> 10 ng / ml </td></tr><tr><td> GDNF </td><td> R &amp; D Systems </td><td> 212-GD-010/CF </td><td> 10 ng / ml </td></tr><tr><td> NT-3 </td><td> R &amp; D Systems </td><td> 267-N3-005/CF </td><td> 10 ng / ml </td></tr></tbody></table> NA = não-aplicável. Tabela 1. PMEF e Mídia para MES cultura de células ou de diferenciação. <table border="1"><tbody><tr><td> &lt;&gt; Nome forte do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Concentração Final </td></tr><tr><td> 0,1% de gelatina </td><td> StemCell Technologies </td><td> 07903 </td><td> NA </td></tr><tr><td> Poli-DL-ornitina </td><td> Sigma-Aldrich </td><td> P0421 </td><td> 0,1 mg / ml em d 2 H2O </td></tr><tr><td> Rato laminina </td><td> Millipore </td><td> CC095 </td><td> 2 ug / ml em PBS </td></tr><tr><td> 0,25% de tripsina / EDTA </td><td> StemCell Technologies </td><td> 07901 </td><td> NA </td></tr><tr><td> Accumax </td><td> Millipore </td><td> SCR006 </td><td> NA </td></tr></tbody></table> NA = não-aplicável. Tabela 2. Reagentes para revestimento e dissociação.

efficient differentiation of mouse embryonic stem cells into motor neurons

Diferenciação direta de células tronco embrionárias (ES), as células em neurônios funcionais motoras representa um recurso promissor para estudar mecanismos da doença, para filtrar compostos de drogas novas, e para desenvolver novas terapias para doenças do neurônio motor, tais como atrofia muscular espinhal (AME) e esclerose lateral amiotrófica ( ALS). Muitos protocolos actuais usar uma combinação de ácido retinóico (RA) e hedgehog sónica (Shh) para diferenciar estaminais embrionárias de rato (MES) em células do motor neurónios 1-4. No entanto, a eficiência de diferenciação de células de MES em neurónios do motor tem apenas um sucesso moderado. Nós desenvolvemos um protocolo de diferenciação de dois passos 5, que melhora significativamente a eficiência de diferenciação em comparação com os protocolos actualmente estabelecidas. O primeiro passo é melhorar o processo de neuralization por adição de noggin e factores de crescimento de fibroblastos (FGFs). Noggin é uma proteína morfogenética do osso antagonista (BMP) e está implicado na indução neural umae acordo com o modelo padrão de neurogênese e resulta na formação de padrão neural anterior 6. FGF sinalização age sinergicamente com Noggin na indução de formação de tecido neural através da promoção de uma identidade posterior neural 7-9. Neste passo, as células foram condicionadas com MES Noggin, bFGF, e FGF-8 durante dois dias para promover a diferenciação para linhagens neurais. O segundo passo é o de induzir a motor especificação neurónio. Noggin / FGFs expostas células foram incubadas com MES RA e um agonista de Shh, agonista Smoothened (SAG), durante mais 5 dias a fim de facilitar a motor geração neurónio. Para monitorizar a diferenciação de neurónios motores em MES, utilizou-se uma linha de células ES derivado de um ratinho transgénico eGFP expressando sob o controlo do promotor de neurónio motor específico Hb9 1. Usando este protocolo robusto, obtivemos 51 ± 0,8% de eficiência diferenciação (n = 3, p &lt;0,01, teste t de Student) 5. Os resultados de imunofluorescênciacoloração revelou que as células GFP + de expressar os marcadores neuronais específicas, a motor Islet-1 e de colina-acetiltransferase (ChAT). Nosso protocolo de diferenciação em duas fases fornece uma forma eficiente de diferenciar as células MES em neurônios motores da coluna vertebral.

Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Desenvolvemos um novo protocolo para melhorar a eficiência de diferenciação in vitro de células-tronco embrionárias em neurônios motores. As células-tronco diferenciadas adquirido neurônios motores apresenta como evidenciado pela expressão de marcadores neuronais e neurônio motor utilizando técnicas de imuno-histoquímica.

Diferenciação eficiente de células-tronco embrionárias em neurônios motores

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

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Research

JoVE Journal

Biology

22.1K visualizações.  Alfred I. duPont Hospital for Children. Desenvolvemos um novo protocolo para melhorar a eficiência de diferenciação in vitro de células-tronco embrionárias em neurônios motores. As células-tronco diferenciadas adquirido neurônios motores apresenta como evidenciado pela expressão de marcadores neuronais e neurônio motor utilizando técnicas de imuno-histoquímica.

Vídeo: Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Biologia

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Na diferenciação in vitro de rato-tronco embrionárias (MES) células usando o método da gota de suspensão

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparação de células embrionárias de camundongos Fibroblastos adequados para a cultura de células-tronco embrionárias humanas e pluripotentes induzidas

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Geração rápida e eficiente de neurônios a partir de células-tronco pluripotentes humanas, em um formato de placa Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Diferenciação de camundongos neonatos pele derivadas de células-tronco em células germinativas semelhantes<em&gt; In vitro</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Retroviral Infecção murina Embryonic Stem Cell Derived Cells embrioides corpo de Análise da diferenciação hematopoética

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...