This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) at the National Institute of Neurological Disorders and Stroke. We would like to thank Dr. Ronald D. McKay for his discussion and comments on this project.

Base de uma única célula não-colônia tipo monocamada (NCM) cultura de hPSCs. 1. Preparações <ol><li> Adicione 500 ml de meio de cultura de fibroblastos de rato embrionários (MEFs): meio DMEM suplementado com 10% de FBS, 2 mM de L-glutamina, 0,1 e aminoácidos não essenciais (NEAA) mM. </li><li> Isolar fibroblastos de rato embrionárias (MEFs) derivadas a partir da estirpe CF1, seguindo um protocolo de rotina 16 e MEFs cultura em 0,1% de 6 cavidades da placa de cultura de células revestidas com gelatina, em meio DMEM. Como alternativa, comprar ações do MEF na passagem 3 a partir de recursos comerciais. </li><li> Prepare placas Matrigel. <ol><li> Dilui-se 5 ml de estoque de Matrigel hESC qualificado com 5 ml de meio DMEM/F12 (arrefecida a ~ 4 ° C) e armazenar até 50% em alíquotas de um congelador de -20 ° C. Descongelar o estoque de Matrigel congelado no frigorífico (~ 4 ° C) O / N e dilui-se ainda mais o Matrigel em meio DMEM/F12 fria (para dar origem a uma concentração de trabalho de 2,5%). </li><li&gt; O revestimento 6 placas de cultura de células com 1,5 ml de 2,5% de Matrigel por poço, armazenar as placas revestidas em frigorífico S / N (Nota: utilizar a placa de Matrigel-revestido dentro de 2 semanas). </li><li> Remova a placa de Matrigel da geladeira, deixe a placa para aquecer à temperatura ambiente na capa de cultura de células de 10 a 30 min (Nota: não aquecer a placa em uma incubadora de cultura de células) e aspirado DMEM meio antes de chapeamento hPSCs. </li></ol></li><li> Adicione 500 ml de meio de células estaminais embrionárias humanas: 80% de meio de DMEM/F12, 20% KSR, 2 mM de L-glutamina, 0,1 mM de aminoácidos não essenciais, 0,1 mM β-mercaptoetanol, e 4 ng / ml de FGF-2. </li><li> Preparar 10 ml de meio de congelação 2x HPSC: 60% de FBS, 20% de DMSO, e 20 uM Y-27632 em meio mTeSR1, esterilizar por filtração, e de usar o meio dentro de 1 semana. </li></ol> 2. Protocolo 1 (Básico): Crescer HPSC colônias em Alimentadores <ol><li> Use números de passagem MEF a 5 ou 6 (designado como p5 e p6) para cultura HPSC, a fim de obter resul consistentets. Mitoticamente inactivar MEFs por tratamento das células com 10 ug / ml de mitomicina C, durante 3 horas a 37 ° C, lavar as células 3x com fosfato tamponado salino da 1X Dulbecco (STP-D), e, em seguida, se dissociam MEFs com 0,05% de tripsina em 0,53 mM EDTA. Alternativamente, irradiar MEFs a uma dose de 8000 rads com um irradiador de raios-X. </li><li> Conte o número de células utilizando o método de exclusão com Trypan Blue mancha sob microscópio. Como alternativa, use um contador automático de células. </li><li> Placa MEFs irradiados em placas de poliestireno de 6 poços revestidos com gelatina a 0,1% a uma densidade de 1,88 x 10 5 células por poço (ou seja, 1,96 x 10 4 células / cm 2). Incubar as células a 37 ° C e 5% de CO 2 durante 24 horas. </li><li> Remova o meio de cultura MEF por aspiração com uma pipeta de Pasteur (Nota: não as etapas de lavagem necessárias neste momento). </li><li> Colônias Triturar HPSC da cultura anterior em pequenos pedaços, examinar os aglomerados sob microscópio para garantir que seus tamanhos que variam from 50 a 100 mM, em diâmetros, e placa aglomerados HPSC sobre o topo das camadas alimentadoras MEF num poço contendo 2 ml de meio HPSC. </li><li> Mude hESC média diária de 3 a 5 dias, a registro o crescimento da colônia por fotografia, marque qualquer colônias morfologicamente alterados ou diferenciadas (~ 5%), e remover manualmente as colônias marcadas pela suavemente aspiração com uma pipeta Pasteur. </li><li> Lavar as colónias remanescentes no MEFs duas vezes (2 min cada com D-PBS), e incuba-se as colónias, com 2 ml de 1 mg / ml de colagenase IV em meio HPSC por 10 a 30 min), e analisar o desprendimento de colónias do HPSC superfície MEF-revestido (Nota: use um raspador para ajudar o processo de desapego somente se as colônias são fortemente ligado à placa). </li><li> Adicionar 5 ml de meio HPSC a cada poço para minimizar reações enzimáticas, colônias de transferência para um tubo de 15 ml, permitir colônias HPSC para sedimentar por 3 a 5 minutos à temperatura ambiente, e garantir a sedimentação de colônias por visualização direta do cells na parte inferior do tubo (Nota: não se centrifugar o tubo nesta fase). </li><li> Retire os sobrenadantes contendo MEFs residuais e dissociado células individuais, voltar a suspender as colônias com 5 ml de meio de células estaminais embrionárias humanas, e repita o passo sedimentação duas vezes. </li><li> Retire as médias, colônias HPSC triturar em pequenos pedaços, loja em 1X HPSC congelamento médio (ou CryoStore CS10 meio de congelamento) para criopreservação (1 confluente bem por frasco congelado) ou placa em uma placa de 6 poços para passaging celular. </li></ol> 3 Protocolo 2:. Converter HPSC Colônias de Alimentadores para NCM <ol><li> Lavar peletes HPSC em D-PBS, uma vez, incuba-se as peletes com 1 ml de 1X Accutase durante 10 min, e examinar a reacção enzimática sob um microscópio para assegurar a dissociação de uma única célula. </li><li> Encerrar as reacções enzimáticas por ressuspensão suave das células em 5 ml de meio contendo 10 uM mTeSR1 Y-27632, seguido por centrifugação a 200 xg à temperatura ambiente durante 5 min(Nota, não use força excessiva de centrifugação, que causam danos às células). </li><li> Adicionar 5 ml de meio mTeSR1 ao sedimento, ressuspender o sedimento de células individuais, e filtro de células dissociadas através de um filtro de células de 40 mM (para remover quaisquer agregados celulares residuais). </li><li> Semente 1,3-2 x 10 6 para um poço hPSCs (1,4-2,1 x 10 5 células / cm 2) de uma placa de 6 poços revestidas com 2,5% de células estaminais embrionárias humanas qualificado Matrigel, adicionar meio mTeSR1 até 2,5 ml, e incluem 10 uM Y-27632 no meio (para facilitar o plaqueamento inicial de 24 horas de uma única célula). Em alternativa, utilizar os seguintes pequenas moléculas para substituir 10 mM Y-27632 para melhorar chapeamento de uma única célula: 1 PM Y-39983 (ROCK I inibidor), 1 phenylbenzodioxane mM (inibidor ROCHA II), 1 PM thiazovivin (um novo inibidor ROCK) , e 2 uM inibidor de JAK 1. </li><li> Substitua o meio com meio mTeSR1 livre de drogas no dia seguinte (dentro de 24 horas), dissociar as células de um adicionalbem, e contagem do número de células para determinar a eficiência de uma única célula de revestimento (Nota: aproximadamente, 50 a 90% de uma única célula plaqueamento eficiência que podem ser alcançados nesta fase). </li><li> Permitir que as células a crescer como uma única formada de células em monocamada durante alguns dias com 3 ml de meio mTeSR1. Mude média diária. </li><li> Empiricamente determina o ritmo de passaging adaptado NCM, dependendo da densidade celular. Passagem, quando o crescimento celular atinge confluência no dia 3 ou dia 4, ou no ponto de tempo de 4 horas após o desaparecimento das fronteiras da célula-a-célula. Dissociar as células em 1 ml de Accutase, utilizar uma proporção de 1 a 3 desdobramento (isto é, uma bem confluente de células plaqueadas em 3 cavidades de placa de 6 poços) para passagens em células e estabilizar adaptado NCM por 5 passagens. </li><li> Congelamento celular <ol><li> Utilizar um bem confluentes (5-6 x 10 ~ 6 células) para um frasco congelado: dissociar as células confluentes de uma bem com 1 mL de Accutase durante 10 min. </li><li> Diluir wom 5 ml de células em meio de centrifugação e mTeSR1 como descritos acima. </li><li> Ressuspender o sedimento em 500 ul de meio mTeSR1 e, em seguida, adiciona-se lentamente 500 ul de 2X HPSC meio de congelação (contendo 20 ^ M Y-27632) em um frasco de criopreservação. Alternativamente, ressuspender as células em 1X HPSC meio de congelamento contendo inibidor 2 uM JAK 1 ou meio 1X CryoStor CS10, na presença de 10 uM ou Y-27632 ou 2 fiM inibidor de JAK 1. </li><li> Colocar os frascos congelados em um cryocontainer gelo-refrigeradas (inferior preenchido com isopranol) e transferir o cryocontainer para um congelador de -80 ° C imediatamente. </li><li> Transferência de células do freezer -80 ° C para um tanque de nitrogênio líquido (no dia seguinte) para a criopreservação de longo prazo. </li></ol></li><li> Descongelamento celular <ol><li> Utilize um frasco de criopreservação para chapeamento um poço de uma placa de 6 poços. </li><li> Meio de pré-aquecer mTeSR1 em um banho de água a 37 ° C durante 10 min, as células descongelamento no mesmo banho de água durante 2 minutos,gota a gota para adicionar as células 5 ml de meio mTeSR1 pré-aquecido contendo 10 uM Y-27632, e centrifuga-se como descrito acima. </li><li> Suavemente ressuspender as pelotas de células com 2 ml de meio contendo 10 uM mTeSR1 Y-27632 e lentamente transferir células de um poço de pré-revestidas com Matrigel (Nota: evitar a produção de bolhas de ar). </li><li> Mude médio diário e esperam que o crescimento HPSC para alcançar confluência no dia 3 ou 4. </li></ol></li></ol> 4 Protocolo 3:. Converter HPSC colônias em Matrigel a NCM Cultura <ol><li> Retirar uma placa revestida de Matrigel do frigorífico, colocar a placa na cultura capa de tecido, durante 10 a 30 minutos, remover o meio de cada poço da placa, e adicionar 2 ml de meio mTeSR1 pré-aquecido a cada poço. </li><li> Transferência triturado aglomerados HPSC da cultura alimentador (Passo 2.10 do protocolo de base) para a placa de Matrigel acima. Adicionar meio mTeSR1 até 2,5 ml de volume final em cada poço. </li><li> Alterar média diária durante 3 a 4 diass até colónias atingir 80 a 90% de confluência. </li><li> Para passagens em células: enxaguar as colónias com D-PBS, duas vezes, o tratamento de células com 1 ml de 2 mg / ml de dispase a 37 ° C durante 15 a 20 min, e de sedimento e de passagem de células como aglomerados. </li><li> Para a cultura NCM: repita os passos 3,1-3,9 definido no protocolo 2. </li></ol> 5 Protocolo 4:. NCM Cultura da hPSCs no LN-521 <ol><li> Descongelar o recombinante LN-521 solução, a 4 ° C, antes do dia da utilização e fazer a solução de revestimento de laminina (LCS), diluindo a laminina descongelado com 1X D-PBS (contendo Ca 2 + / Mg 2 +) para uma concentração final de 10 ug / ml. </li><li> Adicionar 1 ml da LCS a um bem em 6 bem-placa (nota: evitar a seco durante o processo de revestimento). </li><li> Selar a placa revestida com Parafilm para evitar a evaporação e armazenar o prato na geladeira (4 ° C) O / N (nota: use a placa dentro de 1 semana). </li><li> Remova cuidadosamente os LCS com uma pipeta de Pasteur, sem auching a superfície revestida e adicionar 2 ml de meio mTeSR1 para um poço. </li><li> Aquecer todas as soluções de cultura (incluindo D-PBS) em um banho de água a 37 ° C durante 25 min. </li><li> Converta colônias HPSC para NCM <ol><li> Dissociar agregados celulares de células individuais com Accutase conforme descrito no protocolo 2. </li><li> Semente 1,3-2 x 10 6 hPSCs em um LN-521-revestido bem (1,4-2,1 x 10 5 células / cm 2), sem a presença de inibidores de rock. </li><li> Alterar média diária durante 3 a 4 dias. </li><li> Dissociar as células em 1 ml de Accutase no dia 3 ou 4 para a próxima passagem em células usando um 1 a 3 razão de divisão. </li></ol></li></ol> . 6 Protocolo 5: NCM Cultura para plasmídeo DNA transfecção <ol><li> Adaptar colónias HPSC a cultura NCM tal como descrito em Protocolos 2 e 4. </li><li> Placa dissociado hPSCs em 12 poços da placa com uma densidade de células de 7,5 x 10 5 células / poço em meio mTeSR1 na presença de 10 uM Y-27632. </li><li> Substituir médio mTeSR1 com meio mTeSR1 livre de drogas entre 4-8 horas após o plaqueamento das células. </li><li> Para cada transfecção, diluir 2,5 ug de plasmídeos de expressão (por exemplo, pmaxGFP) e 5 ul de Lipofectamina 2000 em tubos Eppendorf separados em 125 ul de cada um de Opti-MEM. </li><li> Após 5 min, misturar os reagentes e incubar diluídos durante 20 min a temperatura ambiente (de modo a formar complexos de transfecção). </li><li> Adicionar os complexos de transfecção 250 ul a cada poço contendo hPSCs em 1 ml de meio mTeSR1 e incuba-se as células a 37 ° C em uma incubadora de CO 2 durante 24 horas. </li><li> Examinar a eficácia de transfecção com um microscópio de fluorescência e fotografia aleatoriamente para o cálculo da eficiência de transfecção real. </li></ol> . 7 Protocolo 6: NCM Cultura para transfecção de MicroRNAs <ol><li> Dissociar hPSCs semi-confluentes no dia 2, em condições NCM, adicionando 1 ml de Accutase por poço em 6<html> Bem-placa. </li><li> Adicionar 10 ml de meio mTeSR1 para diluir as reacções enzimáticas e centrifugar a 200 xg durante 5 min. </li><li> Ressuspender o sedimento de células com meio mTeSR1, semear as células a uma densidade de 7,5 x 10 5 células por poço numa placa de 12 poços em meio contendo 10 uM mTeSR1 Y-27632, e permitir que as células aderir durante 4 h. </li><li> Substitua o meio com meio Y27632-livre mTeSR1. </li><li> Use comercialmente disponível microRNAs (por exemplo, um miRIDIAN miRNA transfecção de controle não-alvo marcado com Dy547). Titula-se concentrações na gama 0-160 nM usando os reagentes fornecidos e seguir as instruções dos fabricantes. </li><li> Optimizar a eficiência de transfecção para cada concentração em linhas HPSC pela imagem da fluorescência Dy547 das células vivas em um microscópio às 24 h após a transfecção. </li><li> Calcular as eficiências de transfecção com base no percentual de células positivas Dy547 sobre todas as células com imagens. </li></ol>. tle &quot;&gt; 8 Protocolo 7: Cultura NCM para a transdução de Lentivirus Vector <ol><li> Repita os passos 7.1 a 7.4 do Protocolo 6. </li><li> Calcular as quantidades de partículas virais a serem utilizadas para a transdução de: usar o seguinte fórmula: TU = (MOI x CN) / VT, enquanto TU indica o número total de unidades de transformação, MOI é igual à multiplicidade desejada de infecção no poço (ou MOI TU / célula), CN especifica o número de células em cada poço, e VT indica os títulos virais de estoque (TU / mL). Por exemplo, uma vez que os títulos virais de imagens SMART-shRNA vector é igual a 1 x 10 9 TU / ml (unidades de transformação por ml), 7,5 x 10 5 células por poço, no tempo de transdução, e esperada uma MOI de 20: 15 de usar mL das partículas virais de ações para cada poço (nota: esta condição é recomendado para indução lentiviral transitória). </li><li> Pré-quentes de 300 ul de meio mTeSR1 com 10 mg / ml de polibreno durante 30 min. </li><li> Adicionar 15 mL de partículas virais de ações emo meio mTeSR1 pré-aquecido contendo polibrene e misture delicadamente a solução. </li><li> Substituir o meio de cultura de células com 300 ul de meio pré-aquecido contendo as partículas virais. </li><li> Após 4 horas de incubação, examinar turboGFP fluorescência (um fabricante de expressão shRNA) para determinar a eficiência de transdução. </li><li> Adicionar 300 mL de meio mTeSR1 adicional para a transdução bem quando as células começam a expressar turboGFP. </li><li> Após 12-16 horas de incubação, reexaminar expressão turbo-GFP. </li><li> Realizar experimentos de acompanhamento desejados com essas células transduzidas dentro de 72 horas. </li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenicity+as+a+clinical+hurdle+for+pluripotent+stem+cell+therapies.">Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies.</a> Nat Med. 19, 998-1004 (2013).</li><li>Domogatskaya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin-511+but+not+-332,+-111,+or+-411+enables+mouse+embryonic+stem+cell+self-renewal+in+vitro.">Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro.</a> Stem Cells. 26, 2800-2809 (2008).</li><li>Domogatskaya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversity+of+laminins.">Functional diversity of laminins.</a> Annu Rev Cell Dev Biol. 28, 523-553 (2012).</li><li>Rodin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+self-renewal+of+human+pluripotent+stem+cells+on+human+recombinant+laminin-511.">Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.</a> Nat Biotechnol. 28, 611-615 (2010).</li><li>Liew, C. 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The authors declare that they have no competing financial interests.

Há duas formas principais de se hPSCs cultura in vitro: cultura convencional do tipo colônia (de células em alimentadores ou matrizes extracelulares) e cultura de suspensão de hPSCs como agregados sem alimentadores 6. As limitações de ambos do tipo colônia e suspensão métodos de cultura incluem heterogeneidade acumulada e mudanças epigenéticas herdadas. Cultura NCM, com base em ambos passaging de uma única célula e plaqueamento de células de alta densidade, representa um novo método pa...

A capacidade de se diferenciar em direção hPSCs tecidos adultos multilinhagem abriu novos caminhos para o tratamento de pacientes que sofrem de doenças graves que envolvem cardiovascular, hepática, pâncreas, e sistemas neurológicos 1-4. Vários tipos de células derivadas de hPSCs também fornecem plataformas celulares robustos para modelagem de doenças, a engenharia genética, a seleção da droga, e 1,4 testes toxicológicos. A questão-chave que garante suas aplicações clínicas e farmacológicas futuras é a geração de um grande número de hPSCs clínica de grau através de cultura in vitro de células. No entanto, sistemas de cultivo atuais são insuficientes ou inerentemente variável, envolvendo várias culturas alimentadoras e sem alimentador de hPSCs como colônias 5,6. Crescimento do tipo colônia de ações hPSCs muitas características estruturais da massa celular interna (ICM) de embriões de mamíferos adiantados. A ICM é propenso a diferenciar-se em três camadas germinativasnum ambiente multicelular, devido à existência de gradientes de sinalização heterogéneos. Assim, a aquisição de heterogeneidade no desenvolvimento embrionário precoce é considerado como um processo necessário para a diferenciação, mas uma característica indesejável de cultura HPSC. A heterogeneidade na cultura HPSC é muitas vezes induzida por sinais apoptóticos excessivas e diferenciação espontânea devido a condições de crescimento abaixo do ideal. Assim, no tipo de colónia de cultura, as células heterogéneas são frequentemente observados na periferia das colónias 7,8. Foi também mostrado que as células em células estaminais embrionárias humanas (células estaminais embrionárias humanas) colónias respostas diferenciais exposição a moléculas de sinalização, tais como BMP-4 9. Além disso, métodos de cultura colônia produzir colheitas de células baixas, bem como as taxas de recuperação muito baixo de células de criopreservação, devido às taxas de crescimento incontrolável e sinalização apoptótica vias 6,9. Nos últimos anos, várias culturas de suspensão têm sido desenvolvidos para hPSCs de cultura, particulArly para a expansão de grandes quantidades de hPSCs dentro e alimentadoras condições livres de matriz 6,10-13. Obviamente, os diferentes sistemas de cultura tem as suas próprias vantagens e desvantagens. Em geral, a natureza heterogénea de hPSCs representa um dos principais inconvenientes da colónia de tipo e de cultura de agregados, que são métodos para a entrega de materiais de sub-óptima de DNA e de RNA em hPSCs por engenharia genética 6. Claramente, há uma necessidade imperiosa de desenvolver novos sistemas que evitam algumas deficiências dos métodos de cultura atual. As descobertas de inibidores de pequenas moléculas (como o inibidor ROCHA Y-27632 e JAK inibidor 1) que melhoram a sobrevivência de uma única célula pavimentar o caminho para a cultura dissociada-HPSC 14,15. Com a utilização destas moléculas pequenas, que desenvolveram recentemente um método de cultura de tipo não-colónia (NCM) crescimento de dissociar-hPSCs 9. Este método de cultura novela combina passaging de uma única célula e de alta densidadechapeamento de métodos, o que nos permite produzir grandes quantidades de hPSCs homogêneas sob ciclos de crescimento consistente, sem grandes anormalidades cromossômicas 9. Alternativamente, a cultura NCM pode ser implementado com diferentes moléculas pequenas e matrizes definidas (como as lamininas), a fim de optimizar o processo de cultura de amplas aplicações. Aqui, apresentamos vários protocolos detalhados com base na cultura NCM e delinear procedimentos detalhados para a engenharia genética. Para demonstrar a versatilidade de protocolos NCM, também testamos cultura NCM com inibidores ROCHA diversas e com a única isoforma laminina 521 (ou seja, LN-521).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Countess automated cell counter&#160;&#160;</td>
			<td style="width:220px;">Invitrogen Inc.&#160;</td>
			<td style="width:187px;">C10227</td>
			<td style="width:187px;">Automatic cell counting</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Faxitron Cabinet X-ray System</td>
			<td style="width:220px;">Faxitron X-ray Corporation, Wheeling, IL&#160;</td>
			<td style="width:187px;">Model RX-650</td>
			<td style="width:187px;">X-ray irradiation of MEFs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">MULTIWELL six-well plates&#160;</td>
			<td style="width:220px;">Becton Dickinson Labware</td>
			<td style="width:187px;">353046</td>
			<td style="width:187px;">Polystyrene plates&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11965&#8211;092</td>
			<td style="width:187px;">For MEF medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">mitomycin C</td>
			<td style="width:220px;">Roche&#160;</td>
			<td style="width:187px;">107 409</td>
			<td style="width:187px;">Mitotic inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Trypsin</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">25300-054</td>
			<td style="width:187px;">For MEF dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM/F12&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11330&#8211;032</td>
			<td style="width:187px;">For hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Opti-MEM I Reduced Serum Medium&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">31985-062</td>
			<td style="width:187px;">For hPSC transfection</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Heat-inactivated FBS</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">16000&#8211;044</td>
			<td style="width:187px;">Component of MEF medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Knockout Serum Replacer&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">10828&#8211;028</td>
			<td style="width:187px;">KSR, Component of hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">14190-144</td>
			<td style="width:187px;">D-PBS, free of Ca2+/Mg2+</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Non-essential amino acids&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">11140&#8211;050</td>
			<td style="width:187px;">NEAA, component of hPSC medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">L-Glutamine&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">25030&#8211;081</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:203px;">mTeSR1 &#38; Supplements</td>
			<td rowspan="2" style="width:220px;">StemCell Technologies</td>
			<td rowspan="2" style="width:187px;">5850</td>
			<td style="width:187px;">Animal protein-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TeSR2 &#38; Supplements</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">5860</td>
			<td style="width:187px;">Xeno-free medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:220px;">Sigma&#160;</td>
			<td style="width:187px;">7522</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;"> 
			MEF (CF-1) ATCC</td>
			<td style="width:220px;">American Type Culture Collection (ATCC)&#160;</td>
			<td style="width:187px;">SCRC-1040</td>
			<td style="width:187px;">For feeder culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">hESC-qualified Matrigel</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">354277</td>
			<td style="width:187px;">For feeder-free culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Laminin-521</td>
			<td style="width:220px;">BioLamina</td>
			<td style="width:187px;">LN521-02</td>
			<td style="width:187px;">Human recombinant protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">FGF-2 (recombinant FGF, basic)</td>
			<td style="width:220px;">R&#38;D Systems, MN</td>
			<td style="width:187px;">223-FB</td>
			<td style="width:187px;">Growth factor in hPSC medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CryoStor CA10&#160;</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">7930</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Accutase</td>
			<td style="width:220px;">Innovative Cell Technologies</td>
			<td style="width:187px;">AT-104</td>
			<td style="width:187px;">1X mixed enzymatic solution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">JAK inhibitor I</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">420099</td>
			<td style="width:187px;">An inhibitor of Janus kinase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">688000</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0012</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-39983</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0029</td>
			<td style="width:187px;">ROCK I inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Phenylbenzodioxane&#160;</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0030</td>
			<td style="width:187px;">ROCK II inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Thiazovivin</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0017</td>
			<td style="width:187px;">A novel ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">BD Falcon Cell Strainer&#160;</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">352340</td>
			<td style="width:187px;">40-&#181;m cell strainer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nalgene 5100-0001 Cryo 1&#176;C</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">C6516F-1</td>
			<td style="width:187px;">&#8220;Mr. Frosty&#8221; Freezing Container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Lipofectamine 2000&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11668-027</td>
			<td style="width:187px;">Transfection reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DharmaFECT Duo&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">T-2010-02</td>
			<td style="width:187px;">Transfection reagent</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">Non-targeting miRIDIAN miRNA Transfection Control</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">IP-004500-01-05</td>
			<td style="width:187px;">Labeled with Dy547, to monitor the delivery of microRNAs&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">SMART-shRNA</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">To be determined</td>
			<td style="width:187px;">Lentiviral vector</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">pmaxGFP</td>
			<td>amaxa Inc (Lonza)</td>
			<td>Included in every transfection kit</td>
			<td style="width:187px;">Expression plasmid for transfection control</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">4-Oct</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-5279</td>
			<td style="width:187px;">Mouse IgG2b, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-1</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21702</td>
			<td style="width:187px;">Mouse IgM, differentiation marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-4</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21704</td>
			<td style="width:187px;">Mouse IgG3, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-60</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21705&#160;</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-81</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21706</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">CK8 (C51)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8020</td>
			<td style="width:187px;">Mouse IgG1, against cytokeratin 8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">&#945;-fetoprotein</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8399</td>
			<td style="width:187px;">AFP, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF-3&#946; (P-19)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-9187</td>
			<td style="width:187px;">FOXA2, goat polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Troponin T (Av-1)</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">MS-295-P0</td>
			<td style="width:187px;">Mouse IgG1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Desmin&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">RB-9014-P1</td>
			<td style="width:187px;">Rabbit IgG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Anti-NANOG</td>
			<td style="width:220px;">ReproCELL Inc, Japan</td>
			<td style="width:187px;">RCAB0004P-F</td>
			<td style="width:187px;">Polyclonal antibody&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Rat anti-GFAP</td>
			<td style="width:220px;">Zymed</td>
			<td style="width:187px;">13-0300</td>
			<td style="width:187px;">Glial fibrillary acidic protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Albumin (clone HSA1/25.1.3)</td>
			<td style="width:220px;">Cedarlane Laboratories Ltd. (</td>
			<td style="width:187px;">CL2513A</td>
			<td style="width:187px;">Mouse IgG1,</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Smooth muscle actin (clone 1A4)</td>
			<td style="width:220px;">DakoCytomation Inc</td>
			<td style="width:187px;">IR611/IS611</td>
			<td style="width:187px;">Mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nestin</td>
			<td style="width:220px;">Chemicon International</td>
			<td style="width:187px;">MAB5326</td>
			<td style="width:187px;">Rabbit polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TUBB3</td>
			<td style="width:220px;">Convance Inc</td>
			<td style="width:187px;">MMS-435P</td>
			<td style="width:187px;">Tuj1, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF4&#945; (C11F12)</td>
			<td style="width:220px;">Cell Signaling Technologies</td>
			<td style="width:187px;">3113</td>
			<td style="width:187px;">Rabbit monoclonal antibody</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Paraformaldehyde (solution)</td>
			<td style="width:220px;">Electron Microscopy Sciences</td>
			<td style="width:187px;">15710</td>
			<td style="width:187px;">PFA, fixative, diluted in D-PBS</td>
		</tr>
	</tbody>
</table>

alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Um esquema geral da cultura NCM A Figura 1 representa um esquema de cultura NCM típico mostrando as alterações dinâmicas de hPSCs depois de alta densidade de revestimento de uma única célula, na presença do inibidor ROCHA Y-27632. Estas alterações morfológicas incluem ligações intercelulares após o plaqueamento, a formação de agregados celulares e crescimento celular exponencial, seguido por condensação de células (Figura 1A).</stro...

Watch this Scientific Journal Video about Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis at JoVE.com

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis | Biology | Video

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para pluripotentes estaminais humanas produção de células, Manutenção e Análise Genética

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

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12.2K visualizações.  National Institutes of Health. Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Vídeo: Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Rato Epidérmico Neural Stem Cell Crest (EPI-NCSC) Culturas

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Biologia

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Cultura e Manutenção de células estaminais embrionárias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparação de pool de plaquetas Lysate Humanos (pHPL) como um suplemento eficiente para animais de soro livre de culturas de células estaminais humanas

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análise de Crescimento Humano embrionárias Colony Stem Cell

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Transfectando e Nucleofecting células-tronco pluripotentes induzidas

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Geração de cardiomiócitos Humanos: Um Protocolo de Diferenciação de Feeder livres Humanos células-tronco pluripotentes induzidas

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente Geração induzida pelo homem células-tronco pluripotentes a partir de células somáticas humanas com Sendai-vírus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Produção automatizada de neurônios cortical e dopaminérgico derivados de células-tronco pluripotentes induzidos por humanos com monitoramento integrado de células vivas

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Uma estratégia de duas etapas que combina modificação epigenética e sinais biomecânicos para gerar células pluripotentes mamíferas

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.