Trabalho descrito neste manuscrito foi possível graças ao financiamento do Instituto de Medicina Regenerativa da Califórnia (CIRM), para UCR Núcleo Stem Cell.

Nosso protocolo começa com um método para se adaptar iPSCs humanos a culturas alimentador livres, seguida de protocolos para a transfecção de iPSCs humanos usando GeneJuice (EMD) e nucleofection de iPSCs humanos usando um dispositivo AMAXA nuclefector. Nota: Os seguintes procedimentos são realizados numa chaminé de fluxo laminar estéril. Todos os meios e soluções são equilibradas a 37 ° C ou à temperatura ambiente antes de iniciar a menos que especificado de outra forma. 1. Estabelecer iPSCs humanos no alimentador-livres IPSCs humanos previamente mantidos em células alimentadoras podem ser divididos e transferidos para Geltrex revestido prato e mantida por duas passagens antes do alimentador sem transfecção. <ol><li> Descongelar Geltrex durante a noite a 4 ° C. Para preparar o revestimento Geltrex, diluir descongelado Geltrex 1:50 em DMEM frio. Misturar as soluções suavemente. </li></ol> Nota: Geltrex, como Matrigel é uma forma solúvel de MATR membrana basalix purificadas a partir de murino Engelbreth-Holm-Swarm (EHS) células tumorais. Alternativamente, Matrigel pode ser usado como uma matriz extracelular de estabelecer alimentador sem culturas humanas IPSC. <ol start="2"><li> Cobrir a totalidade da superfície dos poços de cultura com Geltrex solução (1 ml para um poço de 35 mm). Casaco poços com Geltrex a 37 ° C durante 1 hora incubadora. </li><li> Para iPSCs passagem humanos, adicionar 1 ml de Accutase por poço e incubando a 37 ° C durante 1 min, até a maioria das células começam a separar. </li></ol> Para iPSCs passagem humanos, adicionar 1 ml de Accutase por poço e incubando a 37 ° C durante 1 min, até a maioria das células começam a separar. <ol start="4"><li> Adicionar 10-15 pérolas de vidro para as células e agitar suavemente a placa. Adicionar 2 ml de DMEM KnockOut / F12 e triturar suavemente. Transferir a suspensão de células para um tubo de centrífuga de 10 ml. </li><li> Girar as células a 800 rpm durante 3 minutos à temperatura ambiente. </li><li> Aspirar o sobrenadante dos tubos, deixando humano iPSC pellet intacto. Agite suavemente o tubo paradispersar pellet celular. </li><li> Remover Geltrex do poço revestido. </li><li> Gentilmente ressuspender o pellet em iPSC humano num volume apropriado de STEMPRO. Distribuir entre os poços de alimentadores, dependendo da taxa de proliferação celular). IPSCs humanos podem ser passadas em uma razão de divisão de 1:2 a 1:6. </li><li> Cuidadosamente lugar em 5% CO 2 incubadora, redemoinho a placa cuidadosamente para assegurar uma distribuição uniforme de células em poços. </li><li> Células de alimentação diários até que as células estão prontas para ser dividido de novo (quando as células atingem a confluência de 80%). </li><li> IPSCs passagem em humanos bem novo Geltrex revestido (passo 1,3-1,9) em uma razão de divisão de 1:2. Colónias pequenas devem ser formados e distribuídos uniformemente sobre Geltrex revestido bem antes da transfecção. </li></ol> 2. A transfecção de iPSCs humanos com GeneJuice As células (cultivadas em placas de 6 poços), devem ser, aproximadamente, 40 a 50% confluentes no dia da transfecção para atingir a eficiência de transfecção óptima. Énão é necessário mudar o meio da célula até ao dia seguinte. <ol><li> Preparar 100 uL KO-DMEM/F12 em 1,5 ml de um tubo eppendorf estéril. Adicionar 27 ul de reagente de transfecção GeneJuice. Misturar bem. Incubar à temperatura ambiente durante 5 minutos. </li><li> Adicionar 4 ug de ADN do plasmídeo. Incubar o tubo à temperatura ambiente durante 15 minutos. A escolha do plasmídeo é crítico para a eficiência de transfecção óptima. Usamos um plasmídeo com uma proteína de fluorescência verde melhorada (eGFP) conduzido por promotor CAG 5 (pCAG-eGFP). CAG promotor é um promotor forte que é transcripcionalmente activo em iPSCs humanos e, assim, pode ser utilizado para dirigir a expressão do transgene em tais células. Em nossas mãos, linearização de plasmídeo não parecem afetar a eficiência de transfecção. </li><li> Adicionar GeneJuice DNA-mistura para as células e redemoinho da placa. Girar a placa a 1200 rpm durante 5 minutos (método de &#39;spinoculation&#39;), para aumentar o contacto da mistura de transfecção com iPSCs humanas sobre o poço. </li><li> Incubar as células a 37 &amp;deg; C durante a noite. </li><li> Monitorar a eficácia de transfecção, no dia seguinte. </li><li> Para transfecção estável, alterar médio no dia seguinte com STEMPRO fresco. Adicionar selecção antibiótica apropriada após 24 - 48 horas. </li></ol> 3. Nucleofection de iPSCs humanos IPSCs humanos cultivados em alimentadores ou Geltrex pode ser usado para nucleofection. No entanto, recomendamos repique nucleofected iPSCs humanos para os fibroblastos embrionário (MEF) ou fibroblastos prepúcio humano (HFF) alimentadores para garantir a viabilidade das células de alta e recuperação. Pelo menos 2 milhões de células deve ser utilizado para alcançar a sobrevivência celular após mais nucleofection. <ol><li> Prepare alimentador de células-meio condicionado (MC), incubando KnockOut iPSC soro humano de reposição (KOSR) meio com células alimentadoras durante a noite. Colete CM a cada 24 horas. </li></ol> Nota: Qualquer estirpe de ratinho utilizada para a preparação de camadas alimentadoras (como BLK6, CF1 e MF1) é adequado para ser usado for tornando CM. <ol start="2"><li> No dia da nucleofection, pré-tratamento de humanos com 10 iPSCs inibidor ROCHA uM durante pelo menos 1 hora. </li><li> Preparar 82 ul de solução de Nucleofector humano de células estaminais em 1,5 ml de um tubo eppendorf estéril. Adicionar 18 ul de complemento 1. Misturar bem. Incubar solução a 37 ° C durante 5 mins. </li><li> Pré-aquecimento alimentador célula-condicionado meios de comunicação (CM) e 0,25% de tripsina / EDTA a 37 ° C. Sob o capô, prelabel um tubo estéril 15 ml. </li><li> Remova cuidadosamente a mídia da cultura iPSC humano a ser nucleofected. Lavar suavemente as células com 2 ml de solução de fosfato de 1X tampão (PBS) por poço. Aspirar o PBS. Adicionar 1 ml de tripsina a 0,25% por poço. Incube as células a 37 ° C durante 3 minutos. </li><li> Triturar suavemente as células com 1000 ml ponta da pipeta. Lava-se a parte inferior do poço, para garantir que todas iPSCs humanos são completamente separadas. Transferir a suspensão de células do tubo rotulado 15 ml cónico. </li></ol> Nota: tripsinização em c únicoells deve ser estritamente evitada. As células devem apenas ser desalojada em pequenos aglomerados de células consistiu em aproximadamente tripletos de células. Pequenos aglomerados de células (células triplos) será dissociado em células individuais durante o manuseamento subsequente das células. <ol start="7"><li> Adicionar 9 ml de mídia do MEF para inativar a tripsina. Girar as células a 800 rpm durante 3 minutos. Aspirar cuidadosamente o sobrenadante e deixar o sedimento de células intactas. </li><li> Ressuspender as células em 100 uL de solução pré-aquecida de células estaminais humanas Nucleofector do Passo 3.3. </li><li> Transferir as células para uma cuvete de Nucleofector utilizando uma ponta de pipeta de 1 ml. </li><li> Adicionar 4 ug de DNA do plasmídeo para a suspensão de células na cuvete. Misture células e de DNA por movimentos lentos. Toque a cuvete duas vezes sobre a superfície capuz. </li><li> Coloque a célula no suporte Nucleofector. Usar programas B-016. Células Nucleofect pressionando o botão X. </li></ol> &lt; <ok> &gt; Será exibida quando o processo nucleofection é completed (normalmente leva apenas 1-5 segundos). O uso de outros programas nucleofection (A-023, A-033 e U-023) produziram menos de 10% de células positivas para eGFP de iPSCs humanos. </ok><ol start="12"><li> Recuperar células da cuvete nucleofected usando a pipeta de Pasteur de plástico fornecido. Recuperar células por ressuspensão em CM pré-aquecido e inibidor de rocha em 1,5 ml estéril tubo eppendorf. Incube as células a 37 ° C durante 10 minutos para permitir que as células recuperam. </li><li> Células de transferência de gota a gota, em camadas de alimentação com 1 ml ponteira. Incubar as células a 37 ° C durante a noite. </li><li> Monitorar a eficácia de transfecção, no dia seguinte. Para obter clones estáveis ​​de transgênicos iPSCs humanos que expressam estavelmente transgene, mudar médio no dia seguinte com CM e inibidor ROCK. Adicionar selecção antibiótica apropriada após 24 - 48 horas. </li></ol> 4. Resultados representativos: <img alt="Figura 1" src="/files/ftp_upload/3110/3110fig1.jpg" /> <strong&gt; Figura 1. Fotomicrografias de Riv1 iPSCs humanas transfectadas com pCAG-eGFP. (A) expressam eGFP Riv1 células transfectadas utilizando GeneJuice Geltrex em 12 horas pós-transf ecção. As colónias de Riv1 iPSCs humanos plaqueadas e formado em alimentadores seguintes nucleofection usando B-016 (B) e A-023 (C) do programa. (D) de forma estável que expressam eGFP-humanos colónias iPSC com expressão ubíqua eGFP derivado GeneJuice. (E) estavelmente transfectadas Riv1 iPSCs humanos retido expressão constitutiva eGFP durante a diferenciação do corpo embryoid.

<ol>
	<li>Liew, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells:+possibilities+for+human+cell+transplantation.">Human embryonic stem cells: possibilities for human cell transplantation.</a> Ann Med. 37, 521-532 (2005).</li><li>Eiges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Zwaka, T. P., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogous+recombination+in+human+embryonic+stem+cells.">Homogous recombination in human embryonic stem cells.</a> Nat Biotechnol. 21, 319-321 (2003).</li><li>Siemen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Liew, C. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25, 1521-1528 (2007).</li><li>Xia, X., Ayala, M., Thiede, B. R., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-+and+in+vivo-induced+transgene+expression+in+human+embryonic+stem+cells+and+derivatives.">In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives.</a> Stem Cells. 26, 525-533 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+genetic+manipulation+of+human+embryonic+stem+cells.">Improved genetic manipulation of human embryonic stem cells.</a> Nat Methods. 5, 389-392 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+culture+of+human+embryonic+stem+cells+in+conditioned+medium+for+efficient+genetic+modification.">Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.</a> Nat Protoc. 3, 1435-1443 (2008).</li><li>Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+mediates+high-efficiency+stable+gene+knockdown+and+transgene+expression+in+human+embryonic+stem+cells.">Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells.</a> Stem Cells. 26, 1436-1443 (2008).</li><li>Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenesis+in+human+embryonic+stem+cells+as+a+novel+tool+to+define+the+human+neural+lineage.">BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.</a> Stem Cells. 27, 521-532 (2009).</li></ol>

Não há conflitos de interesse declarados.

Nosso resultado de protocolos de técnicas simples, robusto e altamente reprodutível para introduzir transgenes em iPSCs humanos sem efeitos tóxicos proeminente e morte celular. IPSCs humanos devem ser passadas em pedaços mais pequenos de células (células de 5-10) e plaqueadas em placas de Geltrex em alta densidade (1:2), para assegurar a eficiência de transfecção óptima em numerosas pequenas colónias. Para linhagens de iPSC que são mais propensos a diferenciação e morte celular, maior o número de iPSCs humanos (até 4 X 10 6 células) deve ser usada para uma experiência única nucleofection. Ensaio de transfecção transiente gera um grande número de transgenes que expressam iPSCs humanos dentro de 1 dia. Estavelmente transfectadas clones iPSC geralmente aparecem dentro de 7 dias, e essas colônias transgênicas deve estar pronto para ser escolhido dentro de três semanas. O uso de promotor CAG descrito aqui garante expressão ubíqua de repórter EGFP. Sob tais melhorias, nossos protocolos pode ser alimentado em outras aplicações, incluindo a superexpressão, cindução onditional, derivação de linhas de linhagem específica repórter, shRNA ou siRNA knockdown, a recombinação homóloga do gene alvo e.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> 0,25% de tripsina com EDTA </td><td> Invitrogen </td><td> 25200056 </td><td></td></tr><tr><td> 2-mercaptoetanol </td><td> Invitrogen </td><td> 21985-023 </td><td></td></tr><tr><td> 3 milímetros pérolas de vidro </td><td> Fisher Scientific </td><td> 11-312A </td><td> Pérolas de vidro devem ser lavados com ácido clorídrico (HCl) durante a noite, lavada com hidróxido de sódio (NaOH) e água destilada, e esterilizadas por autoclavagem antes da utilização. </td></tr><tr><td> Célula Accutase reagente dissociação </td><td> Invitrogen </td><td> A11105-01 </td><td></td></tr><tr><td> Factor de crescimento de fibroblastos básico (bFGF) </td><td> Invitrogen </td><td> PHG0263 </td><td></td></tr><tr><td> DMEM (glucose elevada) </td&gt; <td> Lonza </td><td> 12-741 F </td><td></td></tr><tr><td> Soro fetal de vitela </td><td> Invitrogen </td><td> 16000044 </td><td></td></tr><tr><td> Geltrex </td><td> Invitrogen </td><td> 12760013 </td><td> Fator de crescimento reduzido </td></tr><tr><td> GeneJuice reagente de transfecção </td><td> EMD Biosciences </td><td> 70967 </td><td></td></tr><tr><td> Glutamax-I (100X) </td><td> Invitrogen </td><td> 35050061 </td><td> L-glutamina (Invitrogen, 25030081) também podem ser usados ​​em seu lugar. </td></tr><tr><td> Humana iPSC KOSR mídia </td><td></td><td></td><td> 500 ml humano media ES consiste em 390 ml de DMEM KnockOut / F12, 100 ml de soro KnockOut Replacer, 5ml Glutamax-I (100X), NEAA 5ml (100X), 500 ul de 2-mercaptoetanol (55 mm) e suplementado com 10 ng / ml bFGF . </td></tr><tr><td> KnockOut DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td></td></tr><tr><td>Substituição KnockOut Soro (KOSR) </td><td> Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> Rato mídia fibroblastos embrionárias </td><td></td><td></td><td> MEF mídia consiste em 90% de FBS, NEAA 1X, 1X Glutamax-I e 1X piruvato de sódio, em meio DMEM (glucose elevada) </td></tr><tr><td> Aminoácido não essencial, NEAA? (100X) </td><td> Invitrogen </td><td> 11140050 </td><td></td></tr><tr><td> Células estaminais humanas solução Nucleofector um com suplemento </td><td> Lonza </td><td> VAPH-5012 </td><td></td></tr><tr><td> Solução salina tamponada com fosfato sem Ca 2 + e Mg 2 + </td><td> Invitrogen </td><td> 10010023 </td><td></td></tr><tr><td> Piruvato de sódio (100X) </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> STEMPRO meio kit </td><td> Invitrogen </td><td> A1000701 </td><td> 500 ml STEMPRO mídia consiste em 454 ml de KnockFora DMEM / F12, 10 ml STEMPRO suplemento de crescimento isento de soro (50X), 5ml Glutamax-I (100X), 5ml NEAA (100X), 36 ml de BSA (albumina de soro bovino, 25%), 909 ul de 2-mercaptoetanol (55mm ) e suplementado com 10 ng / ml de bFGF. </td></tr></tbody></table>

transfecting and nucleofecting human induced pluripotent stem cells

A modificação genética continua a ser uma ferramenta essencial para o estudo da biologia de células-tronco e em estabelecer potenciais aplicações clínicas de células estaminais embrionárias humanas (hESCs) 1. Embora melhoramentos nos métodos de entrega de vários genes foram descritos 2-9, a transfecção continua a ser um processo caprichoso para hESCs, e ainda não foi relatado em células-tronco pluripotentes induzidas (iPSCs). Neste vídeo, é mostrado como o nosso laboratório de rotina e transfects nucleofects iPSCs humanos utilizando plasmídeo com um verde fluorescente melhorada proteína repórter (eGFP). IPSCs humanos estão adaptados e mantidos como alimentador de culturas isentas de eliminar a possibilidade de a transfecção de células de alimentação e para permitir a selecção eficiente de clones estáveis ​​transgénicas iPSC após a transfecção. Para nucleofection, iPSCs humanos são pré-tratados com inibidor ROCHA 11, tripsinizadas em pequenos aglomerados de células, e novamente plaqueadas em nucleofected alimentadores de alimentador de células condicionadomeio para melhorar a recuperação de células. Transgene-expressam iPSCs humanos pode ser obtida depois de 6 horas. Selecção antibiótico é aplicado após 24 horas e estáveis ​​linhas transgénicas aparecer dentro de 1 semana. Nosso protocolo é robusto e reprodutível para linhagens iPSC sem alterar pluripotência destas células.

Watch this Scientific Journal Video about Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells at JoVE.com

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Apesar dos avanços recentes na modificação genética, transfecção de células estaminais embrionárias humanas (hESCs) continua a ser um processo caprichoso. Para nosso conhecimento, métodos sistemáticos e eficiente para transfectar células-tronco pluripotentes induzidas (iPSCs) não foram relatados. Aqui, descrevemos protocolos robustos de forma eficiente e transfectar nucleofect iPSCs humanos.

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 ...

transfecting-and-nucleofecting-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

21.3K visualizações.  University of California Riverside. Apesar dos avanços recentes na modificação genética, transfecção de células estaminais embrionárias humanas (hESCs) continua a ser um processo caprichoso. Para nosso conhecimento, métodos sistemáticos e eficiente para transfectar células-tronco pluripotentes induzidas (iPSCs) não foram relatados. Aqui, descrevemos protocolos robustos de forma eficiente e transfectar nucleofect iPSCs humanos.

Vídeo: Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfecting células-tronco neurais com o Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Biologia

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Geração de células-tronco pluripotentes induzidas pela reprogramação fibroblastos humanos com o Lentivirus TF Stemgent Humanos Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

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 ...

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

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

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

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

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

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

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Geração de Células-tronco pluripotentes induzidas a partir de sangue periférico usando o STEMCCA Lentivirus Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

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 ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.