The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).

1 Preparação de Meios de Cultura <ol><li> Prepare de fibroblastos de rato embrionário (MEF) forma, combinando o seguinte: 445 ml de DMEM, 50 mL de soro bovino fetal (FBS), e solução de reserva de 5 ml de 100x penicilina / ampicilina. Mantenha o filtro meio esterilizado a 4 ° C por não mais que 14 dias. </li><li> Prepare HPSC meio de cultura através da combinação do seguinte contendo soro: 385 ml de DMEM / F12, 100 ml de soro de substituição nocaute (KSR), 5 ml de 100x não essencial solução solução de estoque de aminoácidos, 5 ml penicilina 100x / estoque ampicilina, 5 ml de 100x mercaptoetanol a solução estoque, e 10 ng / ml de bFGF. Mantenha o filtro meio esterilizado a 4 ° C por não mais que 10 dias. </li><li> Prepare a meio isento de soro mTeSR1 combinando o seguinte: 400 ml de meio basal mTeSR1, 100 ml de suplemento de 5x, e 5 ml de solução de estoque de 100x penicilina / ampicilina. Mantenha meio a 4 ° C por não mais que 10 dias. </li><li> Preparar a solução estoque 10x colagenase IV: pesar 0,5 g de energia colagenase IV, edissolvê-la com 50 ml de DMEM / F12 e filtro de esterilizar. Faça alíquotas e armazená-las à temperatura de -20 ° C durante meses. </li><li> Preparar a solução de gelatina a 0,1%: pesam-se 0,5 g de gelatina (a partir de pele de bovino, de tipo B) de alimentação e dissolvê-lo com água desionizada. Mantenha a solução esterilizada em autoclave à temperatura ambiente durante semanas. </li><li> Preparar meio de diferenciação KSR combinando o seguinte: 410 ml de DMEM, 75 mL KSR, 5 ml 100x solução não-essencial estoque de aminoácidos, 5 ml penicilina 100x / solução estoque ampicilina, 5 ml de solução de estoque 100x mercaptoetanol. Mantenha o filtro meio esterilizado a 4 ° C por não mais que 10 dias. NOTA: KSR varia de lote para lote, o que pode afetar a eficiência de diferenciação. Por isso, é melhor testar vários lotes de KSR para encontrar o melhor para a diferenciação. </li><li> Preparar meio de diferenciação N2 combinando o seguinte: 98 ml de DMEM, 1 ml de suplemento de N2 100x e 1 ml de solução de estoque de 100x penicilina / ampicilina. Mantenha o filtro meio esterilizado a 4° C por não mais que 10 dias. </li><li> Preparar meio de diferenciação B27, combinando o seguinte: 480 ml de meio neurobasal, 10 ml de 50x suplemento B27, 5 ml de solução de estoque de GlutaMax 100x, 100x e 5 ml de solução de reserva de penicilina / ampicilina. Mantenha o filtro meio esterilizado a 4 ° C por não mais que 10 dias. </li></ol> 2 Cultura de hESCs e hiPSCs em células alimentadoras MEF Os hESCs H9 foram obtidos a partir de Instituto de Pesquisa WiCell e hiPSCs foram estabelecidas em laboratório Reijo Pera através de transdução mediada por retrovírus de Yamanaka fatores OCT3 / 4, SOX2, KLF4 e C-MYC 18. <ol><li> Pelo menos um dia antes da passagem de células, preparar algumas placas de gelatina adicionando 1 ml de 0,1% de gelatina a uma cavidade de placas de 6 poços, e incuba-se as placas a 37 ° C durante pelo menos 30 min. </li><li> Após a incubação, a partir de placas de gelatina aspirado, e semente de irradiação inactivado células MEF nas placas a uma densidade de 1,6 x 10 5 células/ Poço em meio de MEF utilizando um hemocitómetro para contar as células. NOTA: Se necessário, preparar alimentadores MEF logo aos três dias antes da passagem da célula. </li><li> Células de passagem um dia após o plaqueamento alimentadores MEF: médias aspirado de hPSCs, adicionar 1 mg / ml de colagenase IV em células em 1 ml / poço, e incuba-se as células a 37 ° C durante 1 hora. </li><li> Durante esta incubação, lavam alimentadores MEF uma vez com PBS e, em seguida adicionar água morna (37 ° C) HPSC meio de cultura contendo soro a 1,5 mL / poço. </li><li> Depois de 1 hora, toque na placa de cultura de um par de vezes para que a maioria das colônias indiferenciadas vai separar as placas, enquanto as colônias diferenciadas permanecem ligados. Cuidadosamente recolher as colônias flutuantes, e transferi-los para tubo de 15 ml. </li><li> Lavar suavemente a placa uma vez com água morna (37 ° C) de DMEM / F12 e transferir DMEM / F12 no mesmo tubo de 15 ml. </li><li> Centrifugar os tubos a 179 xg por 3 min. </li><li> Aspirar médio dos tubos, tomando cuidado para não perturbar o sedimento. Adicionar guerram HPSC meio de cultura, Pipeta as células para cima e para baixo várias vezes para quebrá-las em pequenos aglomerados e misture bem. </li><li> Transferência das células para novos alimentadores MEF em 1: 3-1: 6 ratio. </li><li> Troca da mídia todos os dias e passagem das células a cada 5-6 dias. </li></ol> 3 Preparação de Células para Diferenciação <ol><li> Pelo menos um dia antes da preparação de células, preparar algumas placas de Matrigel (tipicamente três poços de uma placa de 6 poços). Dilui-se com o Matrigel frio (4 ° C) de DMEM / F12 a 1:40 e adicionar 1 mL de Matrigel por poço de placas de 6 poços. Incubar as placas de Matrigel a 4 ° C durante a noite. NOTA: É possível selar as placas de Matrigel com Parafilm e armazená-las a 4 ° C durante o tempo que uma semana. </li><li> Use células (ver Passo 2) 4 dias após a passagem. Remova todas as colônias diferenciadas das placas. Aspirar o meio da placa de cultura, adicionar 1 ml de Accutase por poço e incubar as células a 37 ° C durante 5 min. </li><li> Durante a incubação, preparar alguns gelatin placas por adição de 1 ml de gelatina a um poço de placas de 6 poços. Colocar estas placas e as placas de Matrigel preparadas um dia antes na incubadora. </li><li> Após a 5 min de incubação ou quando as células tornaram-se semi-flutuante, as células de pipeta para cima e para baixo várias vezes para fazê-los em células individuais. </li><li> Recolha de células individuais em tubos de 15 ml e centrifugar a 258 xg durante 3 min. </li><li> Suspenda as células com uma quantidade apropriada de soro contendo meio de cultura, HPSC e adicionar Thiazovivin para a concentração final de 2 fiM. </li><li> Transferência de células em placas revestidas com gelatina na proporção 1: 1, e incubar as células a 37 ° C durante 30 minutos para remover os alimentadores de MEF. </li><li> Transferência das células para um tubo cónico de 15 ml, adicionar soro contendo meio apropriado HPSC e contagem das células com um hemocitómetro. </li><li> Centrifuga-se as células a 258 xg durante 3 min. </li><li> Durante centrífuga, adicionar Thiazovivin se apropriar mTeSR1 meio para fazer uma concentração de 2 uM. </li><li> Depois de centrifugação, umaspirate o meio e adicionar meio mTeSR1 apropriado com Thiazovivin de modo que não são de 1,8 x 10 5 células / ml de meio. </li><li> Retire das placas de Matrigel da incubadora, aspirar o Matrigel e juntar 2 ml de células mistas para um poço das placas de 6 poços. NOTA: A densidade celular deve ser 3,6 x 10 4 células / cm2 de área de superfície. </li><li> Retorne as placas de volta para a incubadora, e deixar que as células anexar, durante 24 horas. </li><li> 24 h após o plaqueamento das células, aspirar meio antigo e adicionar 2 ml de meio novo mTeSR1 por poço e deixar crescer as células durante mais 24 horas antes de iniciar a diferenciação. </li></ol> Diferenciação Celular <ol><li> Iniciar a diferenciação de 48 horas após replating as células em placas de Matrigel. </li><li> Aspirar o meio de cultura da placa, lave as células uma vez com PBS, e adicionar 2 ml do seguinte meio de diferenciação por poço: meio de diferenciação KSR suplementado com 10 pM e 100 nM SB431542 LDN-193189 Marque este dia como D0 de diferenciação. </li><li> Mude meio todos os dias das D0 a D20. NOTA: Pode-se preparar o meio para o uso não mais do que dois dias de cada vez. </li><li> Utilizar o meio seguinte para o D1 e D2: meio de diferenciação KSR suplementado com SB431542 10 mM, 100 nM LDN-193189, 0,25 uM SAG, 2 uM purmorphamine, e 50 ng / ml FGF8b. </li><li> Usar o seguinte meio para a D3 e D4: meio de diferenciação KSR suplementado com SB431542 10 mM, 100 nM LDN-193189, 0,25 uM SAG, 2 uM purmorphamine, 50 ng / ml FGF8b, e 3 uM CHIR99021. </li><li> Utilizar o meio seguinte para D5 e D6: 75% KSR meio de diferenciação, mais 25% de meio de diferenciação de N2 suplementado com 100 nM LDN-193189, 0,25 uM SAG, 2 uM purmorphamine, 50 ng / ml FGF8b, e 3 uM CHIR99021. </li><li> Use o seguinte meio para D7 e D8: 50% KSR meio de diferenciação mais 50% meio de diferenciação N2 suplementado com 100 nM LDN-193189 e 3 mM CHIR99021.</li><li> Use o seguinte meio para D9 e D10: 25% KSR meio de diferenciação mais 75% meio de diferenciação N2 suplementado com 100 nM LDN-193189 e 3 mM CHIR99021. </li><li> Utilizar o meio seguinte para D11 e D12: diferenciação de meio B27 suplementado com 3 uM CHIR99021, 10 ng / mL de BDNF, 10 ng / mL de GDNF, a 1 ng / ml TGF3, ácido ascórbico 0,2 mM e 0,1 mM de cAMP. </li><li> Utilizar o meio seguinte para o resto de diferenciação: meio de diferenciação B27 suplementado com 10 ng / mL de BDNF, 10 ng / mL de GDNF, a 1 ng / ml TGF3, ácido ascórbico 0,2 mM e 0,1 mM de cAMP. </li><li> A cerca de D16 de diferenciação, preparar algumas placas / laminina / Fibronectina poli-L-ornitina. Dilui-se poli-L-ornitina solução estoque com frio (4 ° C) de PBS a 15 ug / ml, adicionar 1 ml de uma cavidade de placas de 6 poços, e incuba-se as placas a 37 ° C durante a noite. </li><li> Aspirar a solução de poli-L-ornitina, lavar as placas 3 vezes com água estéril e depois secar as placas. </li><li> Diluir laminina estoque solbuição com frio (4 ° C) de PBS a 1 mg / ml, adicionar 1 ml de uma cavidade de placas de 6 poços, e incuba-se as placas a 37 ° C durante a noite. </li><li> Aspirar a solução de laminina, lavar as placas 3 vezes com água estéril e depois secar as placas. </li><li> Diluir solução-mãe de fibronectina com frio (4 ° C) de PBS a 2 ug / ml, adicionar 1 ml de uma cavidade de placas de 6 poços, e incuba-se as placas a 37 ° C durante a noite. </li><li> Placas de vedação com Parafilm e colocá-los a 4 ° C por até duas semanas. </li><li> Após 20 dias de diferenciação, replate células para os / as placas Laminin / fibronectina poli-L-ornitina. Aspirar o meio de diferenciação, adicionar 1 ml de água morna (37 ° C) a Accutase um poço de placas de 6 poços, e incuba-se as células a 37 ° C durante 5 min. </li><li> Durante a incubação, colocar as placas / poli-L-ornitina Laminina / fibronectina na incubadora. </li><li> Após a incubação de 5 min ou quando as células tornaram-se semi-flutuante, suavemente as células para cima e para baixo da pipeta várias vezes paratorná-los em células individuais. </li><li> Recolher as células individuais em tubos de 15 mL cónico, e adicionar a quantidade apropriada de meio neurobasal para contagem de células, que irá variar dependendo de expansão (após 11 dias de diferenciação, as células podem expandir de 15-20 vezes). Contar as células usando um hemocitômetro. </li><li> Centrifuga-se as células a 258 xg durante 3 min. Aspirar o sobrenadante e ressuspender as células com meio de diferenciação B27 de modo a que a concentração de células é de 1 x 10 6 células / ml de meio. </li><li> Aspirar fibronectina a partir das placas, lava-se duas vezes com PBS, e adicionar 2 ml de células para um poço de placas de 6 poços. NOTA: A densidade celular para replating deve ser de 2 x 10 5 células / cm2 de área de superfície. </li><li> Mude médio de 24 horas mais tarde para remover as células mortas não acopladas. </li><li> Mudar meio todos os outros dias até que os pontos de tempo desejado para uma dada experiência. </li></ol>

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The authors declare that they have no competing financial interests.

As células estaminais pluripotentes humanas (incluindo ambos os hESCs e hiPSCs) podem ser diferenciadas in vitro para gerar a maioria, se não todos, os tipos de células do nosso corpo, incluindo neurónios DA, o que foi demonstrado em estudos anteriores 19. Aqui, com base no conhecimento do desenvolvimento embrionário neurônio dopaminérgico 20 e protocolos publicados a partir de outros laboratórios 14,15, otimizamos as condições de cultura para a geração de neurônios...

Dopaminérgico (DA) neurônios pode ser encontrada em várias regiões do cérebro, incluindo o mesencéfalo, hipotálamo, retina, e bulbos olfatórios. Os neurônios A9 DA na substância negra pars compacta (SNPC) comportamento de controle e movimento, projetando para o estriado na parte frontal do cérebro e formando o sistema motor extrapiramidal. A degeneração de neurónios DA A9 leva à doença de Parkinson (PD), que é a segunda doença neurodegenerativa mais comum e humano actualmente incurável. A terapia de substituição de células é uma das mais promissoras para o tratamento da DP 1, por conseguinte, tem havido um grande interesse na determinação neurónios A9 DA a partir de células estaminais pluripotentes humanas (hPSCs), incluindo ambas as células estaminais embrionárias humanas (hESCs) e o células humanas recentes tronco pluripotentes induzidas (hiPSCs). Muitas pesquisas têm tentado obter neurônios A9 DA de hPSCs usando vários métodos. Os primeiros relatórios de todos os neurônios DA gerados através de uma neuralroseta estágio progenitor. Por co-cultura com MS5 2 ou PA6 3-5 células estromais com ou sem fatores de crescimento externo para 2-4 semanas, L. Studer e colegas e três outros grupos induzidos com sucesso hESCs para produzir rosetas neurais. Eles, então, enriquecido essas rosetas por dissecção mecânica ou digestão enzimática para uma maior diferenciação. Em outros relatos, os pesquisadores geraram rosetas neurais através do corpo embrióide (EB) flutuantes cultura diferenciação 6-9. Mais tarde, os investigadores estabeleceram um método de diferenciação de monocamada com base 10,11, onde banhado hESCs e hiPSCs na matriz extra-celular, adicionou diferentes factores de crescimento em cultura para induzir a diferenciação de hPSCs de neurónios DA, que imita o in vivo DA desenvolvimento embrionário neurónio . Apesar de todos estes estudos obtidos tirosina hidroxilase (TH) células -expressing com algumas características de neurônios DA, todo o processo de diferenciação é o tempo e trabalho consumindo,geralmente ineficiente, e mais importante ainda, a identidade A9 destes neurónios não foram demonstradas na maioria dos estudos, com excepção de um com Lmx1a expressão ectópica 12. Recentemente, uma placa de novo piso (FP) protocolo baseada foi desenvolvido 13-16, em que os precursores PF com DA potencial neurônio foram gerados primeiro pela ativação do sonic hedgehog e canônicas vias de sinalização Wnt durante a fase inicial de diferenciação, e, em seguida, essas células FP foram melhor especificados para os neurônios de DA. Apesar de este protocolo é mais eficiente, ainda existem alguns problemas; por exemplo, todo o processo de diferenciação leva muito tempo (pelo menos 35 dias) e é dependente de células alimentador 15, ou seja 16 ou EB dependente da identidade A9 não foi demonstrada 14. Aqui, a partir do conhecimento do desenvolvimento in vivo DA neurônio embrionário e os resultados publicados por outros pesquisadores, nós otimizamos as condições de cultivo para o FEPgeração icient de neurônios DA de ambos os hESCs e hiPSCs. Nós primeiro gerado células precursoras FP pela ativação da via de sinalização Wnt canônica com pequena molécula CHIR99021 e sonic hedgehog sinalização com pequenas moléculas SAG e purmorphamine. Estas células FP expressar Foxa2, Lmx1a, CORIN, OTX2 e nestin. Em seguida, essas células especificado PF para neurônios DA com fatores de crescimento, incluindo BDNF, GDNF, etc. Os neurônios DA gerados são do tipo de célula A9 como eles são positivos para GIRK2 enquanto negativo para calbindina 17. Este protocolo é de células de alimentação ou EB independente, altamente eficiente e reprodutível. Usando este protocolo, pode-se derivar neurônios DA em menos de 4 semanas a partir de hESCs ou hiPSCs de pessoas normais para estudo transplante de células, ou de hiPSCs de pacientes com DP para in vitro modelagem de PD ou testar agentes terapêuticos potenciais para a DP.

<table border="0" cellpadding="0" cellspacing="0" width="778">
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			<td height="21" style="height:21px;width:308px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10569-010</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">FBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">26140</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">penicillin/ampicillin</td>
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			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">DMEM/F12</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10565-018</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">KSR</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
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			<td height="21" style="height:21px;width:308px;">Non-essential amino acid</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">b-mercaptoethanol</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">bFGF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB-025</td>
			<td style="width:167px;"></td>
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			<td height="41" style="height:41px;width:308px;">mTesR1</td>
			<td style="width:184px;">STEMCELL Technologies</td>
			<td style="width:119px;">5850</td>
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			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">Gelatin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">N2 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">B27 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">Neurobasal</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">21103-049</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">Glutamax</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:308px;">PBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10010-023</td>
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			<td height="41" style="height:41px;width:308px;">Thiazovivin</td>
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			<td style="width:119px;">sc-361380</td>
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			<td height="21" style="height:21px;width:308px;">SB431542</td>
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			<td height="21" style="height:21px;width:308px;">LDN-193189</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SAG</td>
			<td style="width:184px;">EMD Millipore</td>
			<td style="width:119px;">566660-1MG</td>
			<td style="width:167px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Purmorphamine</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-202785</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FGF8b</td>
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			<td style="width:119px;">C2447-2s</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">BDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">248-BD-025</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">GDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">212-GD-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">TGF-beta3</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">243-B3-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ascorbic acid</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">cAMP</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Mouse anti human NESTIN antibody</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-23927</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human OTX2 antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9566</td>
			<td style="width:167px;">1/2000 dilutiion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Goat anti human FOXA2 antibody</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2400</td>
			<td style="width:167px;">1/200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">rabbit anti human LMX1a antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB10533</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human TH antibody</td>
			<td style="width:184px;">Pel Freez</td>
			<td style="width:119px;">P40101</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Chicken anti human TH antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9702</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mouse anti human TUJ1 antibody</td>
			<td style="width:184px;">Covance</td>
			<td style="width:119px;">MMS-435P</td>
			<td style="width:167px;">1/2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human GIRK2 antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab30738</td>
			<td style="width:167px;">1/300 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human Calbindin antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab25085</td>
			<td style="width:167px;">1/400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Centrifuge</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;">5804</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

directed dopaminergic neuron differentiation from human pluripotent stem cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

Uma visão geral do protocolo de diferenciação é mostrado na Figura 1. A eficiência da diferenciação protocolo aqui apresentada baseia-se no estado das células iniciadoras. Portanto, é essencial ter a certeza de que, em primeiro lugar um remove todas as colónias diferenciadas antes dissociando hPSCs em células individuais de diferenciação, e, segundo, uma esgota a maioria, se não todas, as células alimentadoras MEF por incubação das células em placas revestidas com gelatina durante 30 m...

Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dirigido neurônios dopaminérgicos Diferenciação de Células-Tronco pluripotentes humanas

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

directed-dopaminergic-neuron-differentiation-from-human-pluripotent

Research

JoVE Journal

Neuroscience

16.3K visualizações.  Stanford University School of Medicine. We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson’s disease.

Vídeo: Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. 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 tumorigenicity7-10. 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 problematic11-13. 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 molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivação eficiente dos recursos humanos Progenitores Neuronal e Neurônios de pluripotentes células estaminais embrionárias humanas com a indução de moléculas pequenas

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Neurociência

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

A especificação de telencefálicos neurónios glutamatérgicos de células-tronco pluripotentes humanas

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

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

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

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

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

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

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

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

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

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

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

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Resposta pós-diferenciação de neurônios derivados de células-tronco pluripotentes humanas para triagem de alto conteúdo de Neuritogênese e maturação de sinapse

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Diferenciação neuronal de células-tronco embrionárias do rato in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Derivação, Expansão, Criopreservação e Caracterização de Células Endoteliais Microvasculares Cerebrais de Células-Tronco Pluripotentes Induzidas Humanas

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

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

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

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

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

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

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Células Semelhantes à Microglia Humana: Diferenciação de Células-Tronco Pluripotentes Induzidas e Ensaio de Fagocitose de Células Vivas In Vitro usando Sinaptossomos Humanos

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Culturas organotípicas do córtex humano adulto como modelo ex vivo para transplante e validação de células-tronco humanas

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...