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

Questo lavoro è stato supportato da Alliance L&#39;Atassia di Friedreich ricerca e una borsa di studio pilota da Arnold Family Foundation e il Centro per le cellule staminali e biologia dello sviluppo presso MD Anderson Cancer Center.

1. Virus produzione e trasduzione <ol><li> Plate Phoenix Ampho cellule ad una densità di ~ 7-8x10 6 per piastra 10 cm in 10 ml di terreno DMEM (DMEM elevato glucosio, 10% FBS inattivato al calore, 2 mM L-glutammina, senza antibiotici). Posto nell&#39;incubatrice e coltura di una notte a 37 ° C, 5% CO 2. </li><li> Il giorno seguente trasfezione delle cellule Phoenix con 12 mcg di un vettore codificante sia Oct3 / 4, Sox2, Klf4, c-myc, o gene GFP (Addgene plasmidi 17.217, 17.218, 17.219, 17.220) ...

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Studiare le malattie umane, in particolare neurologiche e neurodegenerative, è stata particolarmente difficile a causa l&#39;inaccessibilità di adeguati modelli cellulari umani. La capacità di riprogrammare facilmente ottenibili cellule somatiche in cellule staminali pluripotenti indotte e la possibilità di differenziarle in tipi di cellule diverse ha aperto la possibilità di creare modelli cellulari di malattie genetiche. Inoltre, iPSCs tenere una grande promessa per il futuro della medicina rigenerativa. Pertanto...

<table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Numero di catalogo </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> Aminoacidi non essenziali </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Butirrato di sodio </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 anticorpi </td><td> Tecnologia delle celle di segnalazione </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60 anticorpo </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 anticorpi </td><td> Tecnologia delle celle di segnalazione </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td> Millipore </td><td> MAB4304 </td></tr><tr><td> CF1 MEF </td><td> Globalstem </td><td> GSC-6201G </td></tr><tr><td> Obiettivo 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-mercaptoetanolo </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> Oggetto 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

Qui presentiamo un protocollo di riprogrammazione fibroblasti umani adulti in cellule pluripotenti indotte umane staminali (hiPSC) utilizzando vettori retrovirali codificanti Oct3 / 4, Sox2, Klf4 e c-myc (OSKM) in presenza di sodio butirrato 1-3. Abbiamo usato questo metodo per riprogrammare il passaggio in ritardo (&gt; p10) fibroblasti umani adulti ottenuti da paziente l&#39;atassia di Friedreich (GM03665, Coriell Repository). L&#39;approccio riprogrammazione comprende protocollo trasduzione altamente efficiente mediante centrifugazione ripetitivo di fibroblasti in presenza di virus contenenti supporti. Le colonie hiPSC riprogrammate sono stati identificati usando immunostaining dal vivo per Tra-1-81, un marcatore di superficie delle cellule pluripotenti, separato da fibroblasti non riprogrammate e manualmente diversi passaggi 4,5. Questi hiPSC state poi trasferite in piastre Matrigel e coltivate in condizioni prive di alimentazione, direttamente dalla piastra riprogrammazione. A partire dal primo passaggio, colonie hiPSC dimostrare caratteristica hES-lmorfologia ike. Usando questo protocollo più del 70% delle colonie selezionati può essere espansa con successo e stabilito in linee cellulari. Le linee stabilite hiPSC visualizzata caratteristici marcatori pluripotenziali tra cui marcatori di superficie TRA-1-60 e SSEA-4, così come marcatori nucleari Oct3 / 4, Sox2 e Nanog. Il protocollo qui presentato è stato stabilito e testato utilizzando fibroblasti ottenuti da pazienti adulti Friedreich atassia e individui di controllo 6, fibroblasti umani neonati, nonché cheratinociti umani.

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 presentiamo un protocollo efficace per la riprogrammazione di cellule somatiche umane in cellule staminali pluripotenti indotte umane (hiPSC) che utilizzano vettori retrovirali codificanti Oct3 / 4, Sox2, Klf4 e c-myc (OSKM) e l&#39;individuazione di hiPSC correttamente riprogrammata mediante colorazione dal vivo con Tra- 1-81 anticorpo.

Selezione e isolamento di colonie di cellule pluripotenti indotte umane staminali riprogrammato da fibroblasti adulti

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

Research

JoVE Journal

Biology

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

Generazione di cellule staminali pluripotenti indotte da fibroblasti umani Riprogrammare con il Stemgent Lentivirus umani TF 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 ...

Ricerca

Biologia

Isolating Stem Cells from Soft Musculoskeletal Tissues

Isolare cellule staminali da tessuti molli muscoloscheletrici

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

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

Trasfettando e Nucleofecting umani cellule staminali pluripotenti indotte

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

Riprogrammare cellule somatiche umane in cellule staminali pluripotenti indotte (iPSCs) Utilizzo vettore retrovirale con 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

Preparazione di cellule di fibroblasti embrionali di topo adatto per colture di cellule pluripotenti umane embrionali e staminali indotta

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

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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

Generazione di cellule pluripotenti indotte umane staminali da sangue periferico mediante il STEMCCA lentivirale Vector

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

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Naïve Adult Stem Cells isolamento da colture primarie di fibroblasti umani

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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

Generazione di cardiomiociti umani: Un protocollo di differenziazione da alimentatore-Free umani cellule staminali pluripotenti indotte

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...