Name: selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts
Uploaded: 2012-02-20T14:00:00
Description: Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

Este trabalho foi apoiado pela Aliança de Friedreich Ataxia Research e uma subvenção piloto da Família Arnold Foundation e O Centro de Células-Tronco e Biologia do Desenvolvimento no MD Anderson Cancer Center.

1. Produção de vírus e de transdução <ol><li> Plate Phoenix células ampho a uma densidade de 8x10 ~ 7-6 por placa de 10 cm em 10 ml de meio DMEM (DMEM glucose elevada, 10% de FBS inactivado calor, 2 mM de L-glutamina, sem antibióticos). Lugar na incubadora e da cultura durante a noite a 37 ° C, 5% de CO 2. </li><li> No dia seguinte, transfectar células Phoenix, utilizando 12 ug de um vector de codificação quer OCT3 / 4, Sox2, KLF4, c-myc, ou GFP gene (Addgene plasmídeos 17.217, 17.218...

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O estudo de doenças humanas, especialmente neurológicas e neurodegenerativas, foi particularmente difícil devido à falta de acesso aos humanos adequados modelos de celulares. A capacidade de reprogramar células somáticas de fácil obtenção em células tronco pluripotentes induzidas e potencial para diferenciá-las em diversos tipos celulares abriu uma possibilidade de criar modelos celulares de doenças genéticas. Além disso, iPSCs realizar uma grande promessa para o futuro da medicina regenerativa. Portanto, ...

<table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11330 </td></tr><tr><td> KSR </td><td> Invitrogen </td><td> 10828 </td></tr><tr><td> Aminoácidos não essenciais </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Butirato de sódio </td><td> Sigma </td><td> B5887 </td></tr><tr><td> Y27632 </td><td> Stemgent </td><td> 04-0012 </td></tr><tr><td> bFGF </td><td> Stemgent </td><td> 03-0002 </td></tr><tr><td> Tra-1-81 anticorpo </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> OCT3 / 4 anticorpo </td><td> Santa Cruz </td><td> sc-8628 </td></tr><tr><td> Nanog anticorpo </td><td> Cell Technology Sinalização </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60 anticorpo </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 anticorpo </td><td> Cell Technology Sinalização </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td> Millipore </td><td> MAB4304 </td></tr><tr><td> CF1 MEFs </td><td> Globalstem </td><td> GSC-6201G </td></tr><tr><td> Marcador objetivo </td><td> Nikon </td><td> MBW10010 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354277 </td></tr><tr><td> mTeSR1 </td><td> StemCell Technologies </td><td> 05850 </td></tr><tr><td> β-mercaptoetanol </td><td> Sigma </td><td> M7522 </td></tr><tr><td> Fugene 6 </td><td> Roche </td><td> 11814443001 </td></tr><tr><td> polibrene </td><td> Sigma </td><td> H9268 </td></tr><tr><td> Marcador de objeto </td><td> Nikon </td><td> MBW10010 </td></tr></tbody></table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

Apresentamos aqui um protocolo de reprogramação fibroblastos humanos adultos em humanos células pluripotentes induzidas estaminais (hiPSC) utilizando vectores retrovirais que codificam OCT3 / 4, Sox2, KLF4 e c-myc (OSKM) na presença de butirato de sódio 1-3. Usamos este método para reprogramar passagem tardia (&gt; p10) fibroblastos humanos adultos provenientes de paciente ataxia de Friedreich (GM03665, Coriell Repository). A abordagem de reprogramação inclui protocolo de transdução altamente eficiente utilizando centrifugação repetitiva de fibroblastos na presença de vírus que contêm meios de comunicação. As colónias hiPSC reprogramadas foram identificadas utilizando imunomarcação vivo para Tra-1-81, um marcador de superfície de células pluripotentes, separados a partir de fibroblastos não-reprogramadas e manualmente passadas 4,5. Estes hiPSC foram então transferidas para placas de Matrigel e cultivadas em condições isentas de alimentação, directamente a partir da placa de reprogramação. A partir da primeira passagem, as colónias hiPSC demonstrar característica HES-lmorfologia ike. Usando este protocolo mais do que 70% das colónias seleccionadas pode com sucesso ser expandido e estabelecido em linhas celulares. As linhas estabelecidas hiPSC exibido característicos marcadores de pluripotência incluindo marcadores de superfície TRA-1-60 e SSEA-4, bem como marcadores nucleares OCT3 / 4, Sox2 e Nanog. O protocolo apresentado aqui tenha sido estabelecida e testadas usando fibroblastos adultos obtidos a partir de pacientes com AF e indivíduos de controlo 6, fibroblastos humanos recém-nascidos, bem como queratinócitos humanos.

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

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

Nós apresentamos um protocolo para a reprogramação eficiente de células somáticas humanas em células-tronco pluripotentes induzidas (hiPSC) usando vetores retrovirais codificando OCT3 / 4, Sox2, KLF4 e c-myc (OSKM) e identificação de hiPSC corretamente reprogramado pela coloração ao vivo com tradi- 1-81 anticorpo.

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 ...

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

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 ...

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