This work was supported by Friedreich&#x2019;s Ataxia Research Alliance and a pilot grant from Arnold Family Foundation and The Center for Stem Cells and Developmental Biology at M.D. Anderson Cancer Center.

1. Virus production and transduction
<ol>
	<li>Plate Phoenix Ampho cells at a density of ~7-8x106 per 10 cm plate in 10 ml of DMEM medium (DMEM high glucose, 10% FBS heat inactivated, 2 mM L-glutamine, no antibiotics). Place in the incubator and culture overnight at 37&deg;C, 5% CO2.</li>
	<li>The next day transfect Phoenix cells using 12 &mu;g of a vector encoding either Oct3/4, Sox2, Klf4, c-myc, or GFP gene (Addgene plasmids 17217, 17218, 17219, 17220) and 35 &mu;l Fugene 6. Prepare transfectio...

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Studying human diseases, especially neurological and neurodegenerative, has been particularly challenging due to the inaccessibility of adequate human cellular models. The ability to reprogram easily obtainable somatic cells into induced pluripotent stem cells and the potential to differentiate them into diverse cell types opened a possibility to create cellular models of genetic diseases. In addition, iPSCs hold a great promise in the future of regenerative medicine. Therefore, it is essential to develop and optimize re...

<table border="1">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalog number </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>Non-essential aminoacids</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 antibody</td>
 <td>Stemgent</td>
 <td>09-0069</td>
 </tr>
 <tr>
 <td>Oct3/4 antibody</td>
 <td>Santa Cruz</td>
 <td>sc-8628</td>
 </tr>
 <tr>
 <td>Nanog antibody</td>
 <td>Cell Signaling Technology</td>
 <td>4903S</td>
 </tr>
 <tr>
 <td>Tra-1-60 antibody</td>
 <td>Millipore</td>
 <td>MAB4360</td>
 </tr>
 <tr>
 <td>Sox2 antibody</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>Objective 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>&beta;-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>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

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

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 present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

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

selecting-isolating-colonies-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

19.8K visualizações.  University of Texas M.D. Anderson Cancer Center. 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.