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

Dit werk werd ondersteund door de ataxie van Friedreich Research Alliance en een pilot subsidie ​​van Arnold Family Foundation en het Centrum voor stamcellen en ontwikkelingsbiologie bij MD Anderson Cancer Center.

1. Virus productie en transductie <ol><li> Plaat Phoenix ampho cellen een dichtheid van 8x10 ~ 7 6 per 10 cm plaat in 10 ml DMEM medium (DMEM hoog glucose, 10% FBS warmte geïnactiveerd, 2 mM L-glutamine, zonder antibiotica). Plaats in de couveuse cultuur nacht bij 37 ° C, 5% CO2. </li><li> De volgende dag transfecteren Phoenix cellen met behulp van 12 ug van een vector die codeert voor een Oct3 / 4, Sox2, Klf4, c-myc, of GFP-gen (Addgene plasmiden 17217, 17218, 17219, 17220) en 35 ul Fugene 6. ...

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Het bestuderen van menselijke ziekten, met name neurologische en neurodegeneratieve, is een bijzondere uitdaging door de ontoegankelijkheid van voldoende menselijke cellulaire modellen. De mogelijkheid om gemakkelijk verkrijgbaar somatische cellen te herprogrammeren tot geïnduceerde pluripotente stamcellen en het potentieel om te differentiëren in verschillende celtypes opende een mogelijkheid om cellulaire modellen van genetische ziekten te creëren. Daarnaast iPSCs bezit zijn van een grote belofte in de toekomst van...

<table border="1"><tbody><tr><td> Reagens </td><td> Vennootschap </td><td> Catalogusnummer </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> Niet-essentiële aminozuren </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Natriumbutyraat </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 antilichaam </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> Oct3 / 4 antilichaam </td><td> Santa Cruz </td><td> sc-8628 </td></tr><tr><td> NANOG antilichaam </td><td> Cell Signaling Technology </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60 antilichaam </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 antilichaam </td><td> Cell Signaling Technology </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> Doelstelling marker </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-mercapto </td><td> Sigma </td><td> M7522 </td></tr><tr><td> Fugene 6 </td><td> Roche </td><td> 11814443001 </td></tr><tr><td> polybreen </td><td> Sigma </td><td> H9268 </td></tr><tr><td> Object marker </td><td> Nikon </td><td> MBW10010 </td></tr></tbody></table>

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

Hierin we een protocol herprogrammering menselijke volwassen fibroblasten in humane geïnduceerde pluripotente cellen (hiPSC) met retrovirale vectoren die coderen Oct3 / 4 Sox2, Klf4 en c-myc (OSKM) in aanwezigheid van natriumbutyraat 1-3. We gebruikten deze methode om laat de passage herprogrammeren (&gt; p10) volwassen mens fibroblasten afgeleid van ataxie van Friedreich patiënt (GM03665, Coriell Repository). De herprogrammering aanpak omvat efficiënte transductie protocol het veelvuldig centrifugeren van fibroblasten in de aanwezigheid van virus-bevattende media. De geprogrammeerd hiPSC kolonies werden geïdentificeerd met levende immunohistochemie voor Tra-1-81 een oppervlak merker pluripotente cellen gescheiden van niet-geherprogrammeerd fibroblasten en handmatig gepasseerd 4,5. Deze hiPSC werden vervolgens overgebracht naar Matrigel platen en gekweekt in feeder-vrije omstandigheden, rechtstreeks van de herprogrammering plaat. Vanaf de eerste passage, hiPSC kolonies te tonen karakteristieke hES-like morfologie. Met deze protocol meer dan 70% van de geselecteerde kolonies met succes kan worden uitgebreid en opgesteld in cellijnen. De gevestigde hiPSC lijnen weergegeven kenmerk pluripotentie markers zoals markers TRA-1-60 SSEA-4, en nucleaire markers Oct3 / 4 Sox2 en NANOG. Het protocol hier gepresenteerd is vastgesteld en getest met behulp van volwassen fibroblasten afkomstig van ataxie van Friedreich patiënten en controle individuen 6, menselijke pasgeboren fibroblasten, maar ook humane keratinocyten.

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

We presenteren een protocol voor een efficiënte herprogrammering van menselijke somatische cellen in het menselijk geïnduceerde pluripotente stamcellen (hiPSC) met behulp van retrovirale vectoren die coderen voor Oct3 / 4, Sox2, Klf4 en c-myc (OSKM) en de identificatie van de opnieuw aangeleerd hiPSC door live kleuring met Tra- 1-81 antilichaam.

Selecteren en isoleren van Koloniën van de mens geïnduceerde pluripotente stamcellen geherprogrammeerd tegen Adult Fibroblasten

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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