We would like to thank Patrick C. Lee, Cyril Ramathal, and Saravanan Karumbayaram (SK) for their assistance in performing the iPSC derivation and characterization experiments; Aaron Cooper for performing the iPSC analysis experiments; Vittorio Sebastiano and Renee A. Reijo Pera for directing the initial reprogramming efforts; SK, William E. Lowry, Jerome A. Zack, and Donald B. Kohn for directing the establishment of the UCLA GMP facilities permitting the conversion and characterization of clinical-grade iPSCs; Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally Obtained Derivatives (CIDMOD) Initiative to generate safe personalized cellular therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc., and the UCLA CTSI Scholar&#8217;s Award to JAB.

NOTA: Este método foi utilizado na pesquisa, publicada no incrédulo et al 10.. 1. Reprogramação Adulto Human dérmica fibroblastos com STEMCCA <ol><li> Fibroblastos dérmicos humanos (adultos Descongelar HUFS), passagem 4 ou inferior, num banho de água a 37 C ° durante 2 min, tomando cuidado para não mergulhar a parte superior do frasco com água. </li><li> Colocar a suspensão de 1 ml de fibroblastos humanos para um frasco cónico de 15 ml e colocar-se lentamente 4 ml de meio de florins padrão, pré-aquecido a 37 ° C, de uma forma gota a gota, para diluir a sulfóxido de dimetilo (DMSO) e suspender novamente as células. </li><li> Centrifuga-se o frasco a 200 xg durante 10 min à temperatura ambiente. Aspirar o sobrenadante e re-suspender as células num volume adequado de meios para criar uma suspensão de 100.000 células / ml e, em seguida, adicionar 2 ml para um poço de uma placa de seis poços, pré-revestidos durante 20 min com 0,2% de gelatina. NOTA: A irmã bem com igual número de células por linha e condição precisa ser semeado for a contagem de células / multiplicidade de infecção de cálculo (MOI). </li><li> Rocha lado da placa para o lado e para trás e para distribuir uniformemente as células. Incubar durante a noite a 37 ° C, 5% de CO 2, num incubador humidificado. </li><li> No dia da transdução, obter a contagem de células (s) a partir da irmã poço (s) de aproximadamente 24 h após o plaqueamento inicial florins. </li><li> Descongelar 2 x 10 8 TU, ou, concentrado lentivírus equivalente (STEMCCA) e diluir a 1 x 10 8 TU / ml em meio HUF. </li><li> Transduzir células em uma relação de 10-MOI em meios de florins suplementado com 8 ug / ml de agente de transfecção. Misturar uniformemente poços transduzidos e incubar durante a noite a 37 ° C, 5% de CO 2, num incubador humidificado. </li><li> Cerca de 24 horas após a transdução de lentivírus, alterar a mídia HUF a célula-tronco pluripotentes humanas (PSC) médio padrão. </li><li> Nos dias 2 a 6, mudar media diária com meio PSC humano. </li><li> No dia 6, dois placa 0,2% placas revestidas com gelatina de 10 cm de1,5-1,75 x 10 6 MEFs derivadas de ratinhos CF1 em media 12 florins. </li><li> No dia 7, tendo o cuidado de não mais do que a centrifugação de 100 x g, temperatura ambiente utilização de tripsina para retirar os fibroblastos reprogramadas dia 7 de um poço da placa de seis poços e passagem a uma razão 1:16, por plaqueamento de todas as células uniformemente entre duas placas de 10 cm, com a mídia PSC humano fresco, que foram semeadas com MEFs no dia anterior. Distribuir os agregados de células num movimento em espiral no sentido horário e no lugar de uma temperatura de 37 ° C, 5% de CO 2 humidificado incubadora durante a noite. </li><li> Cerca de 48 horas após a semeadura das células reprogramadas para MEFs e todos os dias a partir daí, mudar para fora da mídia PSC humanos gastos com meios frescos. </li><li> Após 3 a 4 semanas, escolher colónias individuais com morfologia típica CES como com uma agulha de calibre 21 e subclonar-se em um prato de 24 poços colocando aproximadamente 8 a 10 partes individuais de colónias específicas em poços individuais de uma solução a 0,2% de gelatina-revestido placa de 24 poços de pré-lançamentoed com MEFs derivadas de ratinhos CF1 em meio PSC humano. NOTA: Após a expansão das colônias escolhidas, as colônias têm de ser caracterizado como pluripotentes por formação de corpo embryoid e análise de marcadores de pluripotência expressão ao nível da proteína. </li></ol> 2. Vector Integração Análise Site e STEMCCA Excisão <ol><li> Analisar o local de integração STEMCCA por amplificação linear não restritiva de PCR tal como descrito 10. </li><li> Depois de ensaiar a assegurar uma integração numa área desejada do genoma, consumo STEMCCA fora, aspirando o meio de ESC humano de idade. Em seguida, combinar 45 ul de concentrado de vírus adeno-Cre-PuroR com 8 ug / ml de agente de transfecção em 3 ml de meio de PSC padrão durante 24 horas. </li><li> Após o período de incubação de 24 horas, aspirar o sobrenadante virai misto e lavar as células duas vezes com meio de PSC humanos. Coloque frescos media PSC humanos, juntamente com 2 mg / ml ï¿½puromicina por um período de 5 dias, mudando a mídia sagacidade diáriah meios frescos e antibiótico. </li><li> Após 5 dias, subclone restantes colónias com uma agulha de calibre 21 para 0,2% de poços revestidos com gelatina, em uma placa de 12 poços pré-revestidos com MEFs derivados de CF1mice e expandir conforme detalhado acima. NOTA: Depois de expansão suficiente para se obter uma massa congelada, a conversão em condições isentas de alimentação é necessária. Espere pelo menos 95% de morte colônia de células-tronco, como a maioria das células não vai ser transduzidas com sucesso e vai sucumbir ao antibiótico durante o período de cinco dias de incubação. Embora adeno-Cre-PuroR integra a uma eficiência de 0,001-1% em cromossomas hospedeiros de células infectadas, reexposing uma amostra livre de colónias do factor-a puromicina para garantir 100% de morte celular não irá confirmar a integração ocorreu 13. </li><li> Descongelar uma ampola de matriz de membrana basal pré-aliquotado em gelo, ao longo de um período de aproximadamente 2 horas (as recomendações do fabricante), até que a matriz é um líquido e diluir para uma concentração final de 1:30 em meio basal frio. Cono número desejado de poços numa placa de seis poços com 1 ml por poço. Deixe descansar em temperatura ambiente por 1 hora. </li><li> -Cross hatch (utilizando uma agulha de calibre 18, ou um vidro ou plástico &quot;ponta&quot;), pelo menos, 20 a 30 colónias a partir de um poço revestido previamente com MEFs. Tenha o cuidado de reduzir MEF edificante e transferir de placa. <ol><li> Gerar o dispositivo de tracejado cruzado através de um número de diferentes abordagens, a partir do aquecimento mais complicados, que puxa, e acabamento de uma pipeta de Pasteur de vidro para uma chama, para o simples uso de uma ponta de pipeta de plástico estéril ou, como no nosso caso, 21- e / ou 18 de bitola agulhas. </li></ol></li><li> Aspirar a matriz do poço após incubação revestido. </li><li> Remover pedaços de colônias por raspagem suave da placa de idade, com uma pipeta de 200 mL e coloque cuidadosamente em 3 ml de mídia combinadas frescos 1 na placa revestida com matriz. Incubar durante a noite em uma temperatura de 37 ° C, 5% de CO2 humidificada. Troca de mídia 48 horas após passaging. </li><li> Extrai-se o ADN genómico a partir de pós-hiPSCs excisada e sem alimentação convertidos, em mais de 80% da confluência, utilizando kits de extracção de DNA comercial. </li><li> Analisar por excisão adequada com primers específicos para integrações exógenos do lentivírus STEMCCA: gDNA-Hendo-MycS-forward, 5&#39;-acgagcacaagctcacctct-3 &#39;; gDNA-hWPRE-reverse, 5&#39;-tcagcaaacacagtgcacacc-3 &#39;. Reconstituir primers liofilizados com água PCR-grade para uma solução estoque de 10 mM. </li><li> Executar uma RCP com o protocolo de cinco passos que se segue: desnaturação inicial a 95 ° C durante 3 min; 35 ciclos de cada um dos seguintes: desnaturação a 98 ° C durante 20 seg, emparelhamento de iniciador a 62 ° C durante 15 segundos, e extensão a 72 ° C durante 15 seg; seguido por um único ciclo de extensão final a 72 ° C durante 3 min. </li><li> Adicione um gel de agarose a 3% em peso a 1,5 gramas de agarose e colocando-o em 50 ml de tampão 1x Tris-acetato-EDTA. Micro-ondas até agarose é dissolvido e coloque em mancha gel DNA em pdiluição roper, misturar e dispensar volume adequado em cassete gel para solidificar durante 1 hora à temperatura ambiente. </li><li> Carga 12 uL de produto de PCR misturado com 3 ul de corante de carga em gel de agarose a 3%. Submeter o gel durante 30 min a 80 V. </li><li> Visualizar com luz ultravioleta para a ausência de uma banda que indica a excisão correcta. </li></ol> 3. A determinação quantitativa de Diferenças Gene expressão usando PCR quantitativo de Pré e Pós-hiPSCs excisadas <ol><li> Extrato de RNA total de hiPSCs pós-excisadas e livre-feeder convertidos por meio de kits de extração de RNA comerciais. </li><li> Reverse-transcrever-se a 1 ug de ARN total, utilizando-se kits comerciais, de acordo com as instruções do fabricante. Use ancorado-oligo (dT) 18 e os iniciadores de hexâmeros aleatórios. NOTA: Certifique-se de que o protocolo de PCR é adaptada para transcritos do gene de mais de ou menos de 4 kb. Primers específicos terá de ser feito para i qualquer gene STEMCCA originalmente integradonto. </li><li> Reconstituir o iniciador de PCR liofilizado com água de grau de uma solução estoque de 20? M. </li><li> Use de amostra de 5 ng por 20 ul de reacção que consiste de sonda UPL 10 uM, 2x LightCycler 480 Sondas mestre, e 20 uM iniciadores directos e inversos. </li></ol> 4. A conversão em condições GMP-grade <ol><li> No primeiro dia de transição os hiPSCs pós-extirpado durante a condições GMP grau, fazer uma mistura dos meios de comunicação que consiste em PSC humano combinado de mídia (media um combinado 1) e grau clínico boas práticas de fabrico (BPF) de mídia compatível (media combinados 2 ) a uma razão de 80:20. </li><li> Aspirar fora as velhas mídias combinadas 1 e colocar 3 ml de novos meios de comunicação combinados 1 e 2, pré-aquecido, na proporção 80:20. Mudar meios diariamente durante 3 dias com esta proporção específica. Coloque as células de volta para uma temperatura de 37 ° C, 5% de CO2. </li><li> No dia 4, fazer a mídia combinado 1 e 2 numa proporção de 50:50. Aspirar fora o velho médio e subste com a mídia pré-aquecido, com os meios de comunicação combinados 1 e 2, na proporção 50:50. Mudança de mídia durante 3 dias com essa relação específica. Coloque as células de volta para uma temperatura de 37 ° C, 5% de CO2. </li><li> No dia 7, fazer a media combinado 1 e 2 na proporção 20:80. Aspirar off do meio velho e substitua-o por meio de pré-aquecido, com os meios de comunicação combinados 1 e 2, na proporção 20:80. Mudança de mídia durante 3 dias com essa relação específica. Coloque as células de volta para uma temperatura de 37 ° C, 5% de CO2. </li><li> No dia 10, fazer os meios de comunicação combinados 1 e 2 em um 0: relação de 100. Aspirar off do meio velho e substitua-o por meio de pré-aquecido, com os meios de comunicação combinados 1 e 2 no 0: relação de 100. Mudança de mídia todos os dias na mesma proporção específica. As células estão agora em meio isento de xeno completamente definido. Coloque as células de volta para uma temperatura de 37 ° C, 5% de CO2. NOTA: Durante todo este processo de conversão, de passagens em rotina com uma agulha de calibre 18 que é necessário para manter o tamanho da colónia e densidade adequada. Plano de passaging células a cada 4 a 5 dias com base no tamanho da colónia, a confluência, e diferenciação. </li><li> Uma demão poço numa placa de seis poços com um substrato isento de xeno definido como recomendado pelo fabricante. </li><li> Passagem Mecanicamente confluente bem, já convertido em condições mídia xeno-livre, a partir de uma placa de seis bem com uma agulha de calibre 18. Importante, colocar novos media (mídia combinados 2) sobre as células com 1x inibidor ROCHA, durante 1 hora, antes de passaging a pré-condição da mídia. </li><li> Aspirar a solução de substrato sintético para fora da nova placa que as células estão a ser transferido para. Remova todas as mídias contendo os aglomerados de células-tronco a partir da placa de idade e transferir para a nova placa. </li><li> Coloque as células de volta para uma temperatura de 37 ° C, 5% de CO2 durante 2 dias, com um mínimo de perturbação física (de preferência placas, uma vez dentro da incubadora, não são directamente atingidas de qualquer forma) para permitir a ligação máxima. NOTA: As células exigirá mudanças mídia diárias. Passagin Continuadag cada 4 dias, será importante. Usando uma agulha de calibre 21, de passagem apenas as partes de colônias com morfologia ESC-como ótima (relação núcleo alto). Isso irá garantir seleção para colônias hiPSC adequadas que vai crescer bem e, eventualmente, produzir colônias homogêneas. Tempo total de derivação da ESC como colônias é entre 15-20 dias após a mudança de mídia combinado inicial. </li><li> Congelar para baixo as hiPSCs post extirpado GMP grau pelo primeiro cross-hatch todas as colônias, utilizando uma agulha de calibre 18. Remover todas as partes fora usando uma pipeta de 200 uL e transferir todos os meios contendo as células para um frasco cónico de 15 ml e centrifugar a 200 xg durante 5 min a 4 ° C. </li><li> Enquanto as células são centrifugação, fazer um meio de congelamento que consiste em volumes iguais de meio frio combinadas 2 e meio de congelação, com uma concentração final de DMSO de 7,5%. </li><li> Depois de células são sedimentadas, aspirar off meio de idade e gota a gota, re-suspender o pellet com a mídia de congelamento. Imediatamente transfer para frascos de congelamento e colocar em um freezer -80 ° C. </li></ol> 5. hiPSCs Caracterizar GMP-grade Post-excisadas <ol><li> Para assegurar a expressão apropriada de marcadores de pluripotência pós-conversão, uso de QPCR para quantificar a expressão de marcadores de pluripotência chave (SOX2, OCT4, e NANOG) utilizando os iniciadores listados na Tabela 1 com os mesmos passos como mencionado no passo 3. QPCR metodologias a seguir mesmos passos listados na etapa 3. </li><li> Use a citometria de fluxo para detectar o ácido não-humano siálico Neu5Gc (ácido -glycolylneuraminic N), de acordo com as condições padrão listados no kit como publicado anteriormente 10. </li><li> Lavar suavemente as placas de cultura celular com células, utilizando solução salina tamponada com fosfato 1x (PBS). </li><li> Dissociar colónias usando reagente de dissociação 1x durante 5 min à temperatura ambiente. </li><li> Durante o tempo de incubação de 5 min do passo 5.4, preparar a solução de bloqueio (fornecida emkit), fazendo um 0,5% Agente de bloqueio em PBS gelado. </li><li> Rotular o seguinte conjunto de tubos para análise de citometria de fluxo: Unstained; 4 &#39;, 6-diamidino-2-fenilindole (DAPI) DAPI; Anticorpo de controlo 1: 200; Anticorpo primário 1: 200; Anticorpo secundário 1: 200 de burro anti-IgG de galinha (H + L); MEFs amostra; Células pós-extirpado amostra na matriz; e células em substrato sintético Sample pós-extirpado. </li><li> Gentilmente desalojar agregados celulares a partir da etapa 5.4, adicionando 2 ml de bloquear conteúdos de solução e de transferência para um tubo cônico de 15 ml. Centrifugar a 80 xg durante 5 min a 4 ° C. </li><li> Lavar as células duas vezes com solução de bloqueio e centrifugar tal como indicado no passo 5.7. </li><li> Gentilmente re-suspender aproximadamente 1 x 10 6 células em 100 ul de solução de diluição com o volume correspondente de cada anticorpo bloqueador. Incubar, com agitação suave a 4 ° C durante 1 h. </li><li> Repetir lavagens três vezes como no passo 5.8. </li><li> Re-suspender o pellet celular em 400 mL de diluente Buffer (do kit) com 1: 100 de DAPI para tubos de 2, 6, 7 e 8, conforme listado na etapa 5.6. </li><li> Células tensão através de uma peneira de 40 mM e executado através de citometria de fluxo. </li></ol>

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James A. Byrne (JAB) and Agustin Vega-Crespo receive research funding from Fibrocell Science, Inc. JAB is a scientific consultant for Fibrocell Science, Inc. No other authors have any competing interests to disclose.

Nós descrevemos uma metodologia de derivar hiPSCs livre de fatores e de fazê-los clinicamente relevante, convertendo essas células em condições GMP grau de diferenciação celular downstream em futuras terapêuticas humanas. Embora este protocolo é amplamente aplicável a uma variedade de tipos de células, optou-se por reprogramar fibroblastos dérmicos humanos, devido à facilidade de extracção a partir do paciente e a sua aplicabilidade terapêutica humana personalizados. Uma vez que as limitações forem cor...

Adult human cells have been shown to be capable of undergoing epigenetic remodeling and reprogramming, as a result of lentiviral-based expression of four key transcription factors4,5. An important advancement in the reprogramming field was the use of a single excisable lentiviral stem cell cassette (STEMCCA), which housed all four reprogramming transcription factors that allowed a precise stoichiometric ratio of protein expression6. Additionally, when transduced in specific multiplicity of infection ranges, STEMCCA can lead to predominantly single genomic integration events during the reprogramming process7. The introduction of an excisable version of STEMCCA, which utilizes Cre/loxP technology followed by excision of the reprogramming vector after derivation of the stem cell line, enabled factor-free human induced pluripotent stem cell (hiPSC) lines to be derived8. Additionally, in order to enhance therapeutic applications of hiPSCs, a novel, quick, and readily applicable methodology for good manufacturing practice (GMP)-grade cell line conversion, from xeno-containing to xeno-free conditions, needed to be implemented. Here, we discuss a relevant methodology that more precisely assesses integrated gene expression differences, specifically when integrated into an intron, and clinical-grade cell conversion into putative GMP conditions. 
Previous research has used only relatively insensitive microarray transcriptional analysis to analyze gene expression differences in integrated genes after STEMCCA transduction3,9. Here, we introduce the methodology of sensitive quantitative polymerase chain reaction (PCR) analysis, to further examine integrated gene expression differences. Importantly, current safe-harbor criteria discard hiPSCs that have genes with viral integrations, thus limiting the applicability of these cells for downstream human cellular therapeutics9. We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMP-grade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions10. This provides significant opportunities for the development of future cell reprogramming experiments, which require clinical-grade conditions to maintain human therapeutic relevance. 
These methodologies provide a foundation upon which current safe-harbor criteria may be expanded to include characterized STEMCCA reprogrammed hiPSC lines that maintain a normal gene expression profile after STEMCCA excision from the integrated intron. Also, full conversion into clinical-grade conditions, free from non-human animal antigens, will help to incorporate many more cell types, which have previously been reprogrammed and characterized only in xeno-containing conditions. These methodologies combined, are persuasive grounds for the US Food and Drug Administration (FDA) to consider expanding their limited approval from human embryonic stem cell (ESC)-based therapeutics to hiPSC-based therapeutics11. 
We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions10. Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus. These stem cells then undergo an excision process followed by gene expression characterization. Finally, the hiPSCs are converted over into GMP-grade conditions by a slow conversion methodology.

<table border="0" cellpadding="0" cellspacing="0" width="521">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:135px;">Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:521px;">Media and reagents</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">DMEM/F12 (basal media)</td>
			<td style="width:111px;">Invitrogen (Carlsbad, CA, USA)</td>
			<td style="width:136px;">11330057</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Fetal bovine serum</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">16000044</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:140px;">Minimum essential medium (MEM) non-essential amino acids (NEAA), 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">11140050</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Glutamax, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">PenStrep, penicillin-streptomycin, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Knockout serum replacement</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">10828028</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Trypsin/EDTA, 0.5%</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15400-054</td>
			<td style="width:135px;">Dilute stock out to 0.05% in 1x PBS</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Basic fibroblast growth factor</td>
			<td style="width:111px;">GlobalStem (Rockville, MD, USA)</td>
			<td style="width:136px;">GSR-2001</td>
			<td style="width:135px;">Reconstitute to 10 &#181;g/ml stock in 0.1% bovine serum albumin dissolved in 1x PBS and store at &#8722;80 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">&#946;-mercaptoethanol</td>
			<td style="width:111px;">Millipore (Billerica, MA, USA)</td>
			<td style="width:136px;">ES-007-E</td>
			<td></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Matrigel (basement membrane matrix)</td>
			<td style="width:111px;">BD Biosciences (San Jose, CA, USA)</td>
			<td style="width:136px;">356231</td>
			<td style="width:135px;">Dilute stock Matrigel vial with 10 ml of DMEM/F12 while on ice for a 1:2 dilution. Aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">CELLstart (Synthetic Substrate)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1014201</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Stemmolecule Y27632</td>
			<td style="width:111px;">Stemgent (Cambridge, MA, USA)</td>
			<td style="width:136px;">04-0012-02</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Puromycin</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1113802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">LightCycler 480 Probes Master</td>
			<td style="width:111px;">Roche (Basel, Switzerland)</td>
			<td style="width:136px;">4707494001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">ProFreeze-CDM Medium/freezing medium</td>
			<td style="width:111px;">Lonza (Basel, Switzerland)</td>
			<td style="width:136px;">12-769E</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Dimethyl sulfoxide</td>
			<td style="width:111px;">Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td style="width:136px;">D8418</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">PBS</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">14190-250</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">100 BP DNA Ladder</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15628019</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">SYBR Safe DNA Gel Stain 10000x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">S33102</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;width:140px;">Agarose</td>
			<td style="width:111px;">Bio-Rad Laboratories, Inc. (Hercules, CA, USA)</td>
			<td style="width:136px;">161-3101</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Gelatin, from porcine skin</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">G1890-100G</td>
			<td style="width:135px;">Make stock at 0.2% in PBS, autoclave and store at room temperature</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">mTeSR1</td>
			<td style="width:111px;">StemCell Technologies (Vancouver, BC, Canada)</td>
			<td style="width:136px;">5850</td>
			<td style="width:135px;">Combine Supplement 5X with the basic medium, aliquot and store at 4 &#176;C for up to 2 weeks.&#160;</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Stemedia NutriStem XF/FF Culture Medium</td>
			<td style="width:111px;">Stemgent</td>
			<td style="width:136px;">05-100-1A</td>
			<td style="width:135px;">Thaw at 4 &#176;C O/N, aliquot and store at 4 &#176;C for up to 2 weeks.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Primocin</td>
			<td style="width:111px;">InvivoGen (San Diego, CA, USA)</td>
			<td style="width:136px;">ant-pm-1</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Accutase (Dissociation Reagent)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1110501</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Donkey anti-Chicken IgG AlexaFluor 488</td>
			<td style="width:111px;">Jackson ImmunoResearch (West Grove, PA, USA)</td>
			<td style="width:136px;">703-546-155</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Polybrene/transfection agent</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:136px;">TR-1003-G</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Plasticware</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">12-well plates</td>
			<td style="width:111px;">VWR (West Chester, PA, USA)</td>
			<td style="width:136px;">29442-038</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">6-well plates</td>
			<td style="width:111px;">VWR</td>
			<td style="width:136px;">29442-042</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">10-cm plates</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z688819</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">18-gauge needle</td>
			<td style="width:111px;">Fisher Scientific (Pittsburgh, PA, USA)</td>
			<td style="width:136px;">148265D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">21-gauge needle</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">14-829-10D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Equipment</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD LSRII Flow Cytometer</td>
			<td style="width:111px;">KSystem by Nikon (Tokyo, Japan)</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD FACSDiva Version 6.1.3 Software</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Kits</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">PureLink Genomic DNA Mini Kit</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">K182000</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">KAPA HiFi Hotstart ReadyMix PCR Kit</td>
			<td style="width:111px;">KAPA Biosystems (Wilmington, MA, USA)</td>
			<td style="width:136px;">KK2601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">High Pure RNA Isolation Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">11828665001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:140px;">Transcriptor First Strand cDNA Synthesis Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">4379012001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Sialix anti-Neu5Gc Basic Pack Kit</td>
			<td style="width:111px;">Sialix (Newton, MA, USA)</td>
			<td style="width:136px;">Basic Pack</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 1</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts mTeSR1 and Nutristem</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 2</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts TeSR2 and Nutristem</td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:140px;">HUF Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Dulbecco&#8217;s modified Eagle&#8217;s medium/F12 [DMEM/F12] supplemented with 10% fetal bovine serum, 1x non-essential amino acids, 1x Glutamax, and 1x Primocin</td>
		</tr>
		<tr height="253">
			<td height="253" style="height:253px;width:140px;">Human Pluripotent Stem Cell Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">DMEM/F12 supplemented with 20% knockout serum replacement, 1x Glutamax, 1x non-essential amino acids, 1x Primocin, 1x &#946;-mercaptoethanol, and 10 ng/ml basic fibroblast growth factor</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;">DMEM/F12, Dulbecco&#8217;s modified Eagle&#8217;s medium/F12; PBS, phosphate-buffered saline.</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
	</tbody>
</table>

derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions

Humanos induzida células estaminais pluripotentes (hiPSCs) pode ser gerado com metodologias de reprogramação à base de lentivírus. No entanto, os traços de genes potencialmente oncogénicos restantes em regiões activamente transcritas do genoma, limitar o seu potencial para a utilização em aplicações terapêuticas humanas 1. Além disso, os antigénios não-humanos derivados de reprogramação ou a diferenciação de células estaminais em derivados terapeuticamente relevantes impede as referidas hiPSCs de ser usado num contexto clínico humano 2. Neste vídeo, apresentamos um procedimento para a reprogramação e analisar hiPSCs livre de fator livres de transgenes exógenos. Estes hiPSCs então pode ser analisado para anormalidades da expressão do gene específico do intrão contendo a lentivírus. Esta análise pode ser realizada utilizando reacção em cadeia da polimerase sensível quantitativa (PCR), que tem uma vantagem sobre as técnicas menos sensíveis anteriormente utilizada para detectar diferenças de expressão do gene 3. Conversão completa emboas práticas de fabrico clínico-grade (BPF) condições, permite relevância clínica humana. Nosso protocolo oferece um outro que os critérios de admissibilidade atuais irão se expandir e incluir caracterizados derivados baseados em hiPSC livre de fatores para aplicações terapêuticas para-humanos decorrentes hiPSCs GMP-grade, o que deverá eliminar qualquer risco de imunogenicidade devido aos antígenos não-humanos-forneceu metodologia. Este protocolo é amplamente aplicável a lentivirais reprogramado células de qualquer tipo e não fornece um método reprodutível para a conversão de células reprogramadas em condições GMP-grau.

Apresenta-se um protocolo para derivar hiPSCs livre de factor de grau clínico usando a abordagem baseada reprogramação por lentivírus STEMCCA. A Figura 1A mostra uma imagem representativa de três linhas de pré-excisada hiPSC diferentes, depois de reprogramação com a abordagem STEMCCA sobre uma camada de MEFs. A principal vantagem da abordagem de reprogramação STEMCCA reside no sucesso reprogramação consistente conseguida por vários cientistas, em diferentes grupos de pesquisa e locais. <str...

Watch this Scientific Journal Video about Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions at JoVE.com

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Derivação e Caracterização de um free-Transgene Human Induced Pluripotent Stem Cell Line e Conversão em Definidas condições clínicas de grau

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

derivation-characterization-transgene-free-human-induced-pluripotent

Research

JoVE Journal

Biology

7.8K visualizações.  University of California, Los Angeles (UCLA). We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions. 

Vídeo: Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivação de células estaminais embrionárias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Biologia

Isolation and Derivation of Mouse Embryonic Germinal Cells

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

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

Isolamento e derivação de células embrionárias de rato Germinal

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

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 ...

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

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

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

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

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

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Seleção e isolamento de colônias de células-tronco pluripotentes induzidas a partir de fibroblastos adultos reprogramado

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Reprogramação de células somáticas humanas em células tronco pluripotentes induzidas (iPSCs) usando vetor retroviral com GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

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 ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Diferenciação de alta eficiência de pluripotentes humanas células-tronco para Cardiomyocytes e Caracterização por citometria de fluxo

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

Extração, marcação e purificação de células específicas de linhagem de folículos antrais humanos

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...