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

Dette arbeidet ble støttet av Friedreich sin Ataksi Forskning Alliance og en pilot stipend fra Arnold Family Foundation og The Center for Stem Cells og utviklingsbiologi ved MD Anderson Cancer Center.

1. Virus produksjon og transduksjon <ol><li> Plate Phoenix Ampho cellene ved en tetthet på ~ 7-8x10 6 per 10 cm plate i 10 ml DMEM medium (DMEM høy glukose, 10% FBS varme inaktivert, 2 mm L-glutamin, ingen antibiotika). Plass i kuvøse og kultur over natten ved 37 ° C, 5% CO 2. </li><li> De neste dag transfektere Phoenix celler ved hjelp av 12 mikrogram av en vektor koding enten Oct3 / 4, Sox2, Klf4, c-myc, eller GFP-genet (Addgene 17217 plasmider, 17218, 17219, 17220) og 35 mL Fugene 6. Forbe...

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Studerer menneskelige sykdommer, spesielt nevrologiske og nevrodegenerative, har vært spesielt utfordrende på grunn av utilgjengelighet av tilstrekkelige menneskelige mobilnettet modeller. Muligheten til å omprogrammere lett tilgjengelig somatiske celler til induserte pluripotent stamceller og potensialet for å skille dem i forskjellige celletyper åpnet en mulighet for å lage cellulære modeller av genetiske sykdommer. I tillegg iPSCs holder en meget lovende i fremtiden av regenerativ medisin. Derfor er det viktig...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalognummer </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> Ikke-essensielle fangsten </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Sodium butyrate </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 antistoff </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> Oct3 / 4 antistoff </td><td> Santa Cruz </td><td> sc-8628 </td></tr><tr><td> Nanog antistoff </td><td> Cellesignalisering Teknologi </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60 antistoff </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 antistoff </td><td> Cellesignalisering Teknologi </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> Mål markør </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-mercaptoethanol </td><td> Sigma </td><td> M7522 </td></tr><tr><td> Fugene 6 </td><td> Roche </td><td> 11814443001 </td></tr><tr><td> polybrene </td><td> Sigma </td><td> H9268 </td></tr><tr><td> Objekt markør </td><td> Nikon </td><td> MBW10010 </td></tr></tbody></table>

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

Heri vi presentere en protokoll for omprogrammering voksent menneske fibroblaster inn menneskeskapt påvirkning pluripotent stamceller (hiPSC) bruker retrovirale vektorer som koder Oct3 / 4, Sox2, Klf4 og c-myc (OSKM) i nærvær av natrium butyrate 1-3. Vi brukte denne metoden for å omprogrammere sent passasje (&gt; P10) voksent menneske fibroblaster derivert fra Friedreich er ataksi pasient (GM03665, Coriell Repository). Den omprogrammering tilnærming inkluderer svært effektiv transduksjon protokollen ved hjelp av repeterende sentrifugering av fibroblaster i nærvær av virus-holdige medier. De omprogrammeres hiPSC kolonier ble identifisert ved hjelp av levende farging for Tra-1-81, en ​​overflate markør for pluripotent celler, atskilt fra ikke-omprogrammeres fibroblaster og manuelt passaged 4,5. Disse hiPSC ble deretter overført til Matrigel plater og vokst i mater-frie forhold, direkte fra omprogrammering plate. Fra den første gangen, hiPSC kolonier demonstrere karakteristiske HMS-lIke morfologi. Ved hjelp av denne protokollen mer enn 70% av utvalgte kolonier kan være vellykket utvidet og etablert i cellelinjer. De etablerte hiPSC linjene vises karakteristiske pluripotency markører inkludert overflatevann markører TRA-1-60 og SSEA-4, samt kjernefysiske markører Oct3 / 4, Sox2 og Nanog. Protokollen som presenteres her er etablert og testet med voksne fibroblaster hentet fra Friedreich sine ataksi pasienter og kontroll enkeltpersoner 6, menneskelige nyfødte fibroblaster, samt menneskelige keratinocytter.

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

Vi presenterer en protokoll for effektiv omprogrammering av humane somatiske celler inn i menneskeskapt påvirkning pluripotent stamceller (hiPSC) bruker retrovirale vektorer som koder Oct3 / 4, Sox2, Klf4 og c-myc (OSKM) og identifisering av korrekt omprogrammeres hiPSC av levende farging med Tra- 1-81 antistoff.

Velge og isolere kolonier av menneskeskapte pluripotent stamceller omprogrammert fra voksne Fibroblaster

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

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

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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 ...

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

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

Omprogrammering Menneskelige Somatiske celler i Utførte pluripotent stamceller (iPSCs) Bruke retrovirale Vector med 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 Mice Derived from Induced Pluripotent Stem Cells

Generasjon Mus Utledet fra Utførte pluripotente stamceller

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

Generasjon av menneskeskapte pluripotente stamceller fra perifert blod Bruke 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, ...

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

Generation of Human Cardiomyocytes: En Differensiering Protokoll fra mater-frie menneskeskapte pluripotent stamceller

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

Effektiv Generation Menneskelig indusert pluripotent stamceller fra menneskelige somatiske celler med 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 ...

Bruk av hudbiopsier for å generere pasientavledede podocytter

Generation of Patient-Derived Podocytes from Skin Biopsies

Forfatter Spotlight: Generering av pasientavledede podocytter fra hudbiopsier  

Generering av pasientavledede podocytter fra hudbiopsier

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...