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

Diese Arbeit wurde von Friedreich-Ataxie Research Alliance und einer Pilot-Zuschuss von Arnold Family Foundation und dem Zentrum für Stammzellen und Entwicklungsbiologie am MD Anderson Cancer Center unterstützt.

1. Virus Produktion und Transduktion <ol><li> Tafel Phoenix Ampho-Zellen bei einer Dichte von ~ 7-8x10 6 pro 10 cm Platte in 10 ml DMEM-Medium (DMEM hohe Glucose, 10% Hitze-inaktiviertem FBS, 2 mM L-Glutamin, keine Antibiotika). Platz im Inkubator und Kultur über Nacht bei 37 ° C, 5% CO 2. </li><li> Am nächsten Tag Transfektion Phoenix Zellen unter Verwendung von 12 ug einem Vektor, der entweder Oct3 / 4, Sox2, Klf4, c-myc oder GFP-Gen (Addgene Plasmide 17.217, 17.218, 17.219, 17.220) und 35 u...

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Studieren menschlicher Krankheiten, insbesondere neurologische und neurodegenerative, wurde eine besondere Herausforderung aufgrund der Unzugänglichkeit der angemessene personelle zellulären Modellen. Die Fähigkeit, leicht erhältlich Körperzellen in induzierte pluripotente Stammzellen umprogrammieren und das Potenzial, um sie in verschiedenen Zelltypen differenzieren eröffnet die Möglichkeit, zelluläre Modelle von genetischen Krankheiten zu schaffen. Darüber hinaus halten IPSCs ein großes Versprechen für die ...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog-Nummer </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> Nicht-essentielle Aminosäuren </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Natriumbutyrat </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-Antikörper </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> Oct3 / 4 Antikörper </td><td> Santa Cruz </td><td> SC-8628 </td></tr><tr><td> Nanog Antikörper </td><td> Cell Signaling Technology </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60-Antikörper </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 Antikörper </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> Ziel-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-Mercaptoethanol </td><td> Sigma </td><td> M7522 </td></tr><tr><td> Fugene 6 </td><td> Roche </td><td> 11814443001 </td></tr><tr><td> Polybren </td><td> Sigma </td><td> H9268 </td></tr><tr><td> Objekt-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

Hier präsentieren wir ein Protokoll der Reprogrammierung erwachsener Mensch menschlichen Fibroblasten in induzierte pluripotente Stammzellen (hiPSC) mit retroviralen Vektoren kodieren Oct3 / 4, Sox2, Klf4 und c-Myc (OSKM) in Gegenwart von Natriumbutyrat 1-3. Wir verwendeten diese Methode, um spät Passage neu zu programmieren (&gt; P10) humanen adulten Fibroblasten von Friedreich-Ataxie Patienten abgeleitet (GM03665, Coriell Repository). Die Umprogrammierung beinhaltet hoch effiziente Transduktion Protokoll unter Verwendung von Fibroblasten wiederholte Zentrifugation in Gegenwart von Virus-enthaltenden Medien. Die modifizierte hiPSC Kolonien identifiziert wurden unter Verwendung lebender Immunfärbung für Tra-1-81, ein Oberflächenmarker von pluripotenten Zellen, aus nicht-programmiert Fibroblasten getrennt und manuell passagiert 4,5. Diese hiPSC wurden dann auf Matrigel Platten übertragen und aufgewachsen in Feeder-freien Bedingungen, direkt aus der Umprogrammierung Platte. Ab dem ersten Durchgang, demonstrieren hiPSC charakteristische Kolonien hES-like Morphologie. Unter Verwendung dieses Protokolls mehr als 70% der ausgewählten Kolonien erfolgreich ausgeweitet und festgesetzt werden, in Zelllinien. Die etablierten hiPSC Linien angezeigt charakteristischen Markern für Pluripotenz einschließlich Oberflächenmarker TRA-1-60 und SSEA-4, sowie nukleare Marker Oct3 / 4, Sox2 und Nanog. Das hier vorgestellte Protokoll wurde eingerichtet und getestet mit adulten Fibroblasten von Friedreich-Ataxie Patienten und Kontrollpersonen 6, menschlichen Neugeborenen Fibroblasten, sowie menschliche Keratinozyten erhalten.

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

Wir präsentieren ein Protokoll für die effiziente Reprogrammierung von menschlichen Körperzellen in die menschlichen induzierten pluripotenten Stammzellen (hiPSC) mit retroviralen Vektoren kodieren Oct3 / 4, Sox2, Klf4 und c-Myc (OSKM) und Identifizierung von richtig umprogrammiert hiPSC durch Live-Färbung mit Tra- 1-81 Antikörper ist.

Selektieren und Isolieren Colonies of Human induzierten pluripotenten Stammzellen aus adulten Fibroblasten Umprogrammierte

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

Erzeugung von induzierten pluripotenten Stammzellen durch Reprogrammierung menschlichen Fibroblasten mit dem Stemgent Menschliche 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 ...

Forschung

Biologie

Isolating Stem Cells from Soft Musculoskeletal Tissues

Isolating Stammzellen aus Soft-Gewebe des Bewegungsapparates

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

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

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

Transfektion und Nucleofecting menschlichen induzierten pluripotenten Stammzellen

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

Reprogrammierung menschlichen Körperzellen in induzierte pluripotente Stammzellen (IPSCs) mit retroviralen Vektoren mit 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

Vorbereitung von embryonalen Maus-Fibroblasten-Zellen zum Kultivieren von humanen embryonalen und induzierten pluripotenten Stammzellen

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

Generation der Mäuse aus induzierten pluripotenten Stammzellen

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

Generation of Human induzierten pluripotenten Stammzellen aus dem peripheren Blut mit dem STEMCCA Lentivirusvektor

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

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

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

Generation of Human Cardiomyocytes: Eine Differenzierung Protokoll von Feeder-freien menschlichen induzierten pluripotenten Stammzellen

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