Este trabalho foi apoiado por uma bolsa para pesquisadores avançados da National Science Foundation suíço e através de doações de NYSTEM (C026446; C026447) ea iniciativa de células-tronco Tri-institucional (Fundação Starr).

1. Preparação de Meios de Cultura, pratos revestidos e Manutenção de hPSCs Preparação 1.1 Mídia Nota: Filtro de todos os meios para a esterilização e armazenar a 4 ° C no escuro por até 2 semanas. Números nomes de reagentes, da empresa e catálogo estão listadas na Tabela de Materiais. <ol><li> DMEM/10% de FBS: Combinar 885 mL de DMEM, 100 ml de FBS, 10 ml de Pen / Strep e 5 ml de L-Glutamina. </li><li> HES-médio: Combinar 800 mL de DMEM/F12, KSR 200 ml, 5 ml de L-glutamina, 5 mL de Pen / Strep, 10 ml de solução de MEM essencial mínimo de aminoácidos, 1 ml β-mercaptoetanol. Adiciona-se 10 ng / ml de FGF-2, depois de filtrar o meio. CUIDADO: β-mercaptoetanol é tóxico, evitar a inalação, ingestão e contato com a pele. </li><li> Meio KSR diferenciação: Combinar 820 mL Knockout DMEM, 150 ml KSR, 10 ml de L-Glutamina, 10 ml de Pen / Strep, 10 ml de solução de MEM essencial mínimo de aminoácidos e 1 ml β-mercaptoetanol. </li><li> N2-differentiatino meio: Dissolve-se 12 g do pó em DMEM/F12 980 mL de dH2O, adicionar 1,55 g de glucose, 2 g de bicarbonato de sódio e 100 mg de APO transferrina humana. Misturar 2 mL de dH2O com 25 mg de insulina humana e 40 mL de NaOH 1 N, adicionar a solução dissolvida ao meio. Adicionar 100 ul de di-hidrocloreto de putrescina, selenito de 60 ul, 100 ul de progesterona e trazer o volume até 1 L com dH2O </li></ol> 1.2 Revestimento de placas de cultura <ol><li> Revestimento Matrigel: Thaw 1 ml congelado matrigel alíquota pipetando 19 ml DMEM/F12 sobre a alíquota até que se dissolva. Remover aglomerados fazendo-o passar através de um filtro de células de 40 ^ m e placa 8 ml/10 cm de disco. Incubar os pratos durante 1 hora à temperatura ambiente. Aspirar o matrigel imediatamente antes do plaqueamento das células. Nota: trabalhar rapidamente com matrigel porque podem aglutinar-se a temperaturas acima de 4 ° C. </li><li> Revestimento PO / Lam / FN: Adicionam-se 10 ml de 1x PBS contendo 15 ug / ml de bromidrato de poli-L Ornithin para uma placa de 10 cm. Incubar o prato durante a noite a 37 ° C. Lavar as placas com PBS 1x e depois adicionar 10 ml/10 cm de disco de 1x PBS contendo 2 mg / ml de rato laminina-I e 2 ug / ml de fibronectina. Incubar os pratos durante a noite a 37 ° C. Antes de plaqueamento das células, remova completamente a solução e deixe secar completamente as placas à temperatura ambiente durante 15-20 minutos por pé-los para a cultura parede capô do tecido, sem a tampa. Nota: Os pratos são completamente seco e pronto para o chapeamento de celular quando pode-se ver as estruturas de cristal na superfície (visível a olho nu). As placas podem ser mantidas à temperatura ambiente durante algumas horas a este estado seco. Pratos PO / Lam / FN tem que estar preparado dois dias antes do tempo. Em casos de emergência Lam / FN pode ser incubada por apenas 2-4 horas. No entanto, isso pode arriscar subótimas resultados diferenciação / sobrevivência. </li></ol> 1.3 Manutenção de hPSCs Nota: hPSCs são mantidos em 0,1% de gelatina e rato mitoticamente inactivadas fibroblastos embrionários (MFE) em HES meio suplementado com 10 ng / ml de FGF-2, conforme descrito anteriormente 10,12. As células devem ser divididas a cada 6-8 dias. <ol><li> Revestir uma placa de 10 cm, com 8 ml de gelatina a 0,1% (em 1x PBS sem cálcio ou magnésio), a temperatura ambiente durante 5 min. </li><li> Descongelar MEFs congeladas rapidamente num banho de água a 37 ° C. Adicionar 1 milhão MEFs para 10 ml DMEM/10% de FBS. </li><li> Aspirar a gelatina e placa das células. Incubar a 37 ° C durante pelo menos 6 horas. </li><li> Aspirar o FBS DMEM/10% a partir da placa MEF preparado, lavar placa com 1x PBS uma vez e adicionar 10 ml de HES-meio suplementado com 10 mM Y-27632, dicloridrato. Permitir que o meio para se aquecer a 37 ° C por 20 min. Nota: Para a divisão manual hPSCs uma câmara de fluxo laminar com um microscópio incorporado é usado. No entanto, as células podem ser passadas através de métodos alternativos adequados. </li><li> Dentro de uma câmara de fluxo laminar com um microscópio incorporado, retire colônias separadas usando um levantador de celular e deixá-los flutuar. </li><li> Use umaSeringa de 1 ml para aspirar as colônias flutuantes e despachá-los para o, placa MEF quente fresco. Transferir cerca de um quarto das células para a nova antena. Incubar a 37 ° C. </li><li> Alimente células diariamente com meio HES fresco. </li></ol> 2. Chapeamento de hPSCs para Diferenciação Nota: hPSCs deve ser dividido ou revestida de diferenciação, quando as colônias são grandes, mas ainda tem arestas cortantes com tão pouco como possíveis células diferenciadoras nas suas fronteiras (ver Figura 1B). Quando as células são mantidas através de passagens em manual, as colónias deve ser suficientemente grande para ser facilmente visto pelo olho. Para começar a sentir certo para este ponto do tempo pode-se manter um prato HPSC separado para duas semanas sem passaging e assistir as células alcançar e passar o ponto de tempo ideal para passaging / diferenciá-los. <ol><li> Preparar placas de 10 cm com matrigel 1 h antes de iniciar a diferenciação. Revestimento matrigel bem sucedida pode serverificada com ampliação de 4x. </li><li> Quando os hPSCs está pronto para ser dividida, aspirar o meio e adicionar 4 ml de 0,05% de tripsina-EDTA para as células. </li><li> Agite o prato na horizontal por 2 min vigorosamente até que se possa ver os MEFs como células individuais levantando a placa sob o microscópio. As colônias HPSC permanecer anexado como colônias. </li><li> Imediata e cuidadosamente, aspirar a tripsina e deixar o prato ficar à temperatura ambiente durante 2-4 min. </li><li> Adicionar 2 ml de HES-meio para a placa e com uma pipeta P1000, separar as células. Nota: Se as células não podem ser levantados facilmente para fora da superfície, a placa pode ser incubada vazio à TA por mais 2-3 minutos. </li><li> Transferir as células a 8 mL de HES-meio suplementado com 10 mM Y-27632, dicloridrato. </li><li> Aspirar o matrigel a partir de uma previamente preparada placa de 10 cm. Placa as células em uma relação de 1:1 ou 1:2 (por exemplo: as células de uma placa de 10 cm são divididos em duas placas de 10 cm) sobre a placa de matrigel. Incubar a 37 °C durante a noite. Nota: Esta camada deve resultar em cerca de 100.000 células / cm 2. </li></ol> 3. Indução da diferenciação neural Nota: A diferenciação pode ser iniciado (dia 0), quando as células são 90-100% confluentes (ver Figura 1C), normalmente no dia seguinte. Se a confluência exata ainda não foi alcançado, as células podem ser alimentados diariamente com HES-médio até que estejam prontos para a diferenciação. Alternativamente, o número inicial de células plaqueadas pode ser aumentada. <ol><li> No dia 0-3, as células alimentar diariamente, com 10 ml/10 cm de disco médio KSR diferenciação contendo 0,1 uM e 10 uM LDN193189 SB431542. </li><li> Nos dias 4 e 5 de alimentação das células com 75% de meio KSR-diferenciação e 25% de meio N2-diferenciação tanto contendo LDN193189 e SB431542. </li><li> No dia 6 e 7 de alimentos para as células com 50% de médio KSR-diferenciação e 50% médio N2-diferenciação ambos contendo LDN193189 e SB431542. </li><li> No dia 8 e 9 de alimentação das células com 25% de meio KSR-diferenciação e 75% de meio N2-diferenciação tanto contendo LDN193189 e SB431542. </li><li> No dia 9 e 10 de preparar pratos PO / Lam / FN, tal como indicado em 1.2.2 por replating das células no dia 11. </li><li> No dia 10 células de alimentação com meio N2-diferenciação contendo LDN193189 e SB431542. </li></ol> 4. Repique em gotas para Especificação NC <ol><li> No dia 11 aspirado Lam / FN a partir dos pratos previamente preparados e deixá-las secar completamente à temperatura ambiente, durante 20-30 minutos, tal como explicado em 1.2.2. </li><li> Remova o meio das células em diferenciação, lave as células uma vez com PBS 1x e adicionar 8 mL accutase/10 cm prato. Incubar durante 20 min a 37 ° C. </li><li> Usando um levantador de celular, separar as células e ressuspender-los em Accutase com uma pipeta de 5 ml. Transferir a suspensão para um tubo de 15 ml. </li><li> Adicionar 5 ml de PBS 1x e girar as células durante 5 min a 114 x g. </li><li> Ressuspender as células em 10 ml de 1x PBS. Para garantir uma suspensão única célula, filtrá-los através de um filtro de células 40 mM e contar as células usando um hemocitômetro ou técnica equivalente. </li><li> Rodar as células para baixo e ressuspender-los em meio N2-diferenciação contendo 200 ^ M do ácido ascórbico (AA), 20 ng / ml de BDNF, 100 ng / ml FGF8, 20 ng / mL de SHH, 10 uM Y-27632, dicloridrato, na concentração de 100 mil -150,000 células/10 jil. </li><li> Usando um repeat-pipeta, placa 10 gotas ul próximos uns dos outros, sem lhes tocar na PO secos / Lam / FN 15 centímetros pratos. Nota: Uma placa de 10 cm de células diferenciadas, normalmente resulta em cerca de 2-3 vezes 15 centímetros de pratos ou de aproximadamente 100 x 10 cm ul droplets/15 prato. </li><li> Deixe as gotículas repousar à temperatura ambiente durante 20-30 minutos, em seguida, muito cuidadosamente (para não perturbar as gotas) adicionar 30 ml de N2/AA/BDNF/FGF8/SHH/Y e incubar a 37 ° C. </li><li> No dia 12, cuidadosamente alimentar as células com 20 ml/15 cm de disco N2/AA/BDNF/FGF8/SHH. </li><li> A partir day 14-17 alimentar as células a cada segundo ou terceiro dia com N2/AA/BDNF/FGF8. </li><li> No dia 16 e 17 de preparar pratos PO / Lam / FN para replate células NC após FACS classificação. </li></ol> 5. Fluorescência celular ativado (FACS) de células NC Nota: A preparação das células para FACS requer cerca de 2 horas. <ol><li> No dia 18 de meio de remoção, lave as células uma vez com PBS 1x no prato e adicionar 12 ml de Accutase. Incubar as células durante 20 min a 37 ° C. </li><li> Usando um levantador de célula, separar as células da superfície e deixá-los flutuar. Pipetar as células em suspensão usando uma pipeta de 5 ml, transferi-los para um tubo de 50 ml e adicionar 30 ml de PBS 1x. Rodar as células durante 5 min a 114 x g. </li><li> Usando uma pipeta P1000, ressuspender as células em 1 ml de 2% de FBS / HBSS (HBSS contém 15 mM de HEPES). Adicionar 19 ml de 2% de FBS / HBSS e filtrar as células através de um coador de células de 40 mM para assegurar uma suspensão de células individuais. Incubar as células em gelo durante 15 min para bloqueio de anticorpos e depois contá-los usando um hemocitômetro. </li><li> Rodar as células para baixo e ressuspender deles na concentração de 10 milhões de células / ml em 2% de FBS / HBSS. </li><li> Separe 1 milhão de células cada um para o imaculado, single-coradas (HNK-1 apenas e só p75) e anticorpo secundário manchado apenas (APC só e 488 apenas) FACS controla. </li><li> Adicionar 5 mL de HNK-1 e 5 uL de anticorpo primário p75 por 10 milhões de células em 1 ml de suspensão apropriados para as amostras e incuba-se durante 20 min em gelo. </li><li> Lavar as células em 10 ml de PBS 1X (5 min de centrifugação a 114 xg) e ressuspender as células em 1 ml de 2% de FBS / HBSS. Adicionar 2 mL de APC e 1 ml de 488 anticorpo secundário por 10 milhões de células em 1 ml de suspensão apropriados para as amostras e incuba-se durante 20 minutos em gelo no escuro. Nota: A APC e 488 são os anticorpos secundários utilizados aqui para a coloração HNK-1 e p75, respectivamente. </li><li> Lave as células em 10 ml de 1x PBS, duas vezes, ressuspender 10 milhões de células em 500 ul de2% de FBS / HBSS contendo DAPI (a 0,5 ng / mL) ou alternativo da mancha de células vivas adequada para excluir as células mortas da análise de FACS. </li><li> Transferir as amostras para tubos de FACS apropriar. </li><li> Prepare os tubos de FACS com 0,5 ml de 2% FBS / HBSS para coleta de células. Nota: As células e tubos de coleta são mantidos em gelo, no escuro, durante o tempo de classificação. </li><li> Usando uma máquina de classificação de células com lasers que podem detectar DAPI, APC e 488 tipo DAPI-/HNK-1 + / P75 + células positivas duplas. Nota: o tempo e manipulação das células Preparação leva a uma pequena percentagem de morte celular, DAPI manchas células mortas e, assim, permite que apenas a estas células a ser excluídos da população classificada. Qualquer mancha vivo / morto alternativa funciona igualmente bem para esta finalidade. O próprio DAPI não provocar citotoxicidade na população de células vivas. </li></ol> 6. Repique de células classificadas, NC Manutenção e Expansão Nota: células FACS classificadas deve ser mãolevou com cuidado especial para garantir a sobrevivência ideal. Mantenha-os no gelo até repique eles. Não vórtice ou pipeta-los duramente. As células podem ser ressuspendidas sacudindo o tubo. <ol><li> Após separação FACS, contar as células de CN, em seguida, colocá-los para baixo e ressuspender em meio N2-diferenciação suplementado com 10 ng / mL de FGF2, 20 ng / mL de EGF e 10 uM dicloridrato Y-27632 no volume apropriado para replate los em 30.000 células / 10 gotas ul. </li><li> Completamente seco previamente preparado PO / placas Lam / FN ea placa de aproximadamente 50-70 gotas por 10 centímetros prato. Deixe os pratos repousar à temperatura ambiente durante 20-30 min. </li><li> Adicionar muito cuidado 20 ml/10 cm prato de N2/FGF2/EGF/Y-27632 sem perturbar as gotas. Incubar as células a 37 ° C. </li><li> Alimentar as células a cada 2-3 dias com N2/FGF2/EGF. Nota: células NC são expandidos ou repicadas aproximadamente a cada 4-5 dias ou quando eles começam a se acumulando dentro das gotículas. </li><li> Para a passagem das células NC, retire o meio, wash as células uma vez com PBS 1x e adicionar 8 mL accutase/10 cm prato. Incubar durante 20 min a 37 ° C. </li><li> Pipetar as células para fora da placa com uma pipeta de 5 ml, e transferi-los para um tubo de 15 ml. Adicionar 6 ml de 1x PBS. </li><li> Rodar as células durante 5 min a 114 x g. </li><li> Ressuspender as células em meio N2-diferenciação suplementadas com 10 ng / mL de FGF2, 20 ng / mL de EGF e 10 uM Y-27632, dicloridrato, a fim de ter 20000 células/10 ul gota. </li><li> Replate as células em 10 gotas ul em placas de PO / Lam / FN secas. Deixe os pratos repousar à temperatura ambiente durante 20-30 min. </li><li> Adicione cuidadosamente 20 ml/10 cm prato de N2/FGF2/EGF/Y-27632. Incubar a 37 ° C. Nota: células NC pode ser mantido ou ampliado por até 2 semanas, como uma população progenitor relativamente homogênea. Cultura já pode levá-los a se diferenciar em vários tipos de células descendentes. </li></ol>

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Para a diferenciação de células bem sucedida R-NC a partir de células estaminais embrionárias humanas / hiPSCs as seguintes considerações devem ser feitas. É crítico para o trabalho sob condições de cultura estéril em todos os momentos. Em particular, é importante testar culturas HPSC de contaminação por micoplasma regular, uma vez que esta contaminação irá dificultar a diferenciação com êxito, mas não pode ser facilmente detectada visualmente em culturas HPSC. A diferenciação de R-NC deve ser in...

Células estaminais embrionárias humanas (hESC) e células-tronco pluripotentes induzidas pelo homem (hiPSC) mostraram um imenso potencial, em particular para a investigação e tratamento futuro de doenças humanas para as quais nem bons modelos animais nem tecidos primários estão disponíveis. Exemplos de aplicação para a tecnologia de células estaminais embrionárias humanas / hiPSC são os seguintes: Células de interesse particular podem ser gerados a partir de células estaminais embrionárias humanas / hiPSCs para a medicina regenerativa em quantidade ilimitada 1. As células podem ser produzidos a partir de pacientes portadores de uma doença específica e utilizado para estabelecer, em modelos de doenças in vitro, 2,3. Tais modelos de doenças pode ser empregado para a triagem de drogas em grande escala na busca de novos compostos de drogas 4, bem como testes de drogas existentes para a eficácia e toxicidade 5. Em modelos de doenças in vitro podem levar à identificação dos mecanismos da doença da novela. Para todas as aplicações da tecnologia de células estaminais embrionárias humanas / iPSC é importante trabalhar com específica, bem definirtipos d célula afetada na doença de interesse. Assim, a disponibilidade de protocolos de diferenciação in vitro sólidos e reprodutíveis é fundamental para todas as aplicações da tecnologia de células estaminais embrionárias humanas / hiPSC. Protocolos são desejáveis ​​que mostram variabilidade mínima, custa tempo, esforço, dificuldade e custo, bem como a reprodutibilidade máxima entre linhas hESC / hiPSC e diferentes pesquisadores. Crista neural (NF), as células surgem durante neurulação vertebrado entre a epiderme e o epitélio neural. Eles proliferam e migram extensivamente durante todo o desenvolvimento do embrião, dando origem a uma diversidade impressionante de tipos de células descendentes, incluindo o osso / cartilagem, o esqueleto craniofacial, nervos sensoriais, as células de Schwann, os melanócitos, células musculares lisas, neurônios entéricos, os neurônios autonômicos, células cromafins , células do septo cardíaco, dentes e adrenal / tireóide células glandulares 6. Assim, células de CN são um tipo de célula atraente para o campo de células-tronco e importante para oa modelagem de uma variedade de doenças, tais como a doença de Hirschsprung, 7, disautonomia familiar 8, bem como os cancros, tais como o neuroblastoma 9. Além disso, eles oferecem a possibilidade de estudar os aspectos do desenvolvimento embrionário humano in vitro. A atualmente disponíveis e amplamente aplicado em protocolo de diferenciação in vitro para a derivação de células da CN de hESCs 10,11 requer até 35 dias de diferenciação e que envolve indução neural em células alimentadoras, como as células do estroma MS5 e é, portanto, realizadas em condições mal definidas. Embora possa ser em maior escala para gerar grandes quantidades de células da CN, por exemplo necessário para high-throughput screening de drogas 4, este é o trabalho e onerosa. Além disso, envolve passaging manual das rosetas neurais, que podem ser difíceis de reproduzir e, portanto, é sujeito a variabilidade global, em particular quando é aplicado a uma grande variedade de células estaminais embrionárias humanas, ou hiPSClinhas. Aqui, a derivação gradual de células NC em um protocolo de 18 dias que está livre de células alimentadoras é mostrado. Este método é mais curto e mais definido do que o protocolo usado atualmente. Além disso, ele é muito robusto na geração de células NC entre diferentes linhas hiPSC. Importante, é mostrado que as células NC gerados por ambos os protocolos surgem na fronteira de rosetas neurais (doravante denominado roseta-NC ou R-NC). As células derivadas utilizando um dos dois protocolos olhar morfologicamente idênticos, eles expressam os mesmos marcadores NC e se agrupam em análise de microarray. Células NC derivadas usando o novo protocolo (R-NC) são funcionais, semelhantes às células NC derivados usando o protocolo de idade (MS5-R-NC) de tal forma que eles podem migrar e diferenciar-se em neurónios mais. Portanto, as células podem ser utilizadas simultaneamente com as células MS5-R-NC. O protocolo de células R-NC para a derivação de células a partir de células estaminais embrionárias humanas NC / iPSC será útil para todas as aplicações da tecnologia hESC / iPSC envolvendo a linhagem NC. &lt;/ P&gt;

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			<td height="21" style="height:21px;width:353px;">Regents</td>
			<td style="width:219px;">company</td>
			<td style="width:160px;">cataloge number</td>
			<td style="width:165px;">comments</td>
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			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12</td>
			<td>Gibco-Life Technologies</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout Serum Replacement</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td>Lot should be tested</td>
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			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Penicinlin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM minimum essential amino acids solution&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-050</td>
			<td></td>
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			<td height="20" style="height:20px;">&#946;-Mercaptoethanol&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>toxic</td>
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			<td height="20" style="height:20px;">Recombinant human FGF basic (FGF2)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
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			<td height="20" style="height:20px;">DMEM/F12 powder&#160;</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
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			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APO human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
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			<td height="20" style="height:20px;">Human insulin&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Putriscine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-L Ornithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dispase in Hank&#39;s Balanced Salt Solution 5U/ml</td>
			<td>Stem Cell Technologies</td>
			<td>7013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BDNF</td>
			<td>R&#38;D Systems</td>
			<td>248-BD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FGF8</td>
			<td>R&#38;D Systems</td>
			<td>423-F8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse recombinant sonic hedgehog (SHH)</td>
			<td>R&#38;D Systems</td>
			<td>464SH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco-Life Technologies</td>
			<td>14170-112</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Gibco-Life Technologies</td>
			<td>1563-080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant EGF</td>
			<td>R&#38;D Systems</td>
			<td>236EG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti HNK-1/N-Cam (CD57) mIgM</td>
			<td>Sigma</td>
			<td>C6680-100TST</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-p75 mIgG1 (Nerve Growth factor receptor)</td>
			<td>Advanced Targeting Systems</td>
			<td>AB-N07</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APC rat anti-mIgM</td>
			<td>BD Parmingen</td>
			<td>550676</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 488 goat anti-mouse IgG1&#160;</td>
			<td>Invitrogen</td>
			<td>A21121</td>
			<td>lot should be tested</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Anti Oct4 mIgG2b (used at 1:200 dilution)</td>
			<td>Santa Cruz</td>
			<td>sc-5279</td>
			<td>lot should be tested</td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Material/Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-6105M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture dishes: 10cm and 15 cm plates, centrifuge tubes, FACS tubes,&#160; pipetts, pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass hematocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture incubator (CO2, humidity and temperature controlled)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture laminar flow hood with embedded microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture biosafety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell sorting machine, i.e. MoFlo</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml TB syringe 27Gx1/2</td>
			<td>BD Biosciences</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell lifter Polyethylene</td>
			<td>Corning Incorporated</td>
			<td>3008</td>
			<td></td>
		</tr>
	</tbody>
</table>

feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells

Células-tronco pluripotentes humanas (hPSCs) têm um grande potencial para o estudo do desenvolvimento embrionário humano, para a modelagem de doenças humanas no prato e como fonte de células para transplante para aplicações regenerativas após doença ou acidentes. Células da crista neural (NF) são os precursores para uma grande variedade de células somáticas adultas, tais como células do sistema nervoso periférico e as células gliais, os melanócitos e células mesenquimais. Eles são uma valiosa fonte de células para estudar aspectos do desenvolvimento embrionário humano, incluindo a especificação destino e migração celular. Além disso a diferenciação de células progenitoras NC em tipos de células diferenciados terminalmente oferece a possibilidade de modelar doenças humanas in vitro, investigar os mecanismos da doença e para gerar células medicina regenerativa. Este artigo apresenta a adaptação de um protocolo atualmente disponível em vitro de diferenciação para a derivação de células a partir de NC hPSCs. Este novo protocolo requer 18 dias de diferentiation, é, facilmente escalável e altamente reprodutível entre (hiPSC) linhas humana (hESC) linhas de células estaminais embrionárias, bem como humana induzida de células-tronco pluripotentes sem alimentador. Ambos os antigos e novos protocolos de produzir células NC de igual identidade.

As duas melhorias mais importantes do protocolo R-NC sobre o protocolo MS5-R NC-11, são as condições de diferenciação definidos isentos de alimentação e a redução global do requisito de tempo. Células alimentadoras MS5 13 são da medula óssea células estromais derivadas de ratinho que foram mostrados para apoiar a diferenciação neural de hESCs 14. HESCs cultivadas em células alimentadoras MS5 em estruturas de baixa densidade forma epiteliais e rosetas neurais 15,</sup...

Watch this Scientific Journal Video about Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells at JoVE.com

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

Crista neural (CN), as células derivadas de células-tronco pluripotentes humanas (HPSC) tem um grande potencial para a modelagem de desenvolvimento humano e de doenças e para terapias de substituição celular. Aqui, uma adaptação livre de alimentador do atualmente utilizado<em&gt; In vitro</em&gt; Protocolo de diferenciação para a derivação de células da CN de hPSCs é apresentado.

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 ...

feeder-free-derivation-neural-crest-progenitor-cells-from-human

Research

JoVE Journal

Neuroscience

14.1K visualizações.  Sloan-Kettering Institute for Cancer Research. Crista neural (CN), as células derivadas de células-tronco pluripotentes humanas (HPSC) tem um grande potencial para a modelagem de desenvolvimento humano e de doenças e para terapias de substituição celular. Aqui, uma adaptação livre de alimentador do atualmente utilizado In vitro Protocolo de diferenciação para a derivação de células da CN de hPSCs é apresentado.

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

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

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

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

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

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

Neurociência

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

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

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

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

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

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

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Isolamento e Cultura de células da crista neural-tronco de folículos capilares humanos

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

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 ...

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

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

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

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

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

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 ...

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.

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 ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Derivação de adultos fibroblastos humanos e sua conversão direta em Expansível Neural células progenitoras

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identificando marcadores de superfície celular da haste neural primária e células progenitoras por rotulagem metabólica de Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...