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  4. Generation of human induced pluripotent stem cells using non-synthetic mRNA
 
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2016
Journal Article
Title

Generation of human induced pluripotent stem cells using non-synthetic mRNA

Abstract
Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods.The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-g) or alpha (hIFN-a) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay.Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.
Author(s)
Rohani, Leili
Fraunhofer-Institut für Zelltherapie und Immunologie IZI  
Fabian, Claire  
Fraunhofer-Institut für Zelltherapie und Immunologie IZI  
Holland, Heidrun
Universität Leipzig
Naaldijk, Yahaira
Fraunhofer-Institut für Zelltherapie und Immunologie IZI  
Dressel, Ralf
Universitätsmedizin Göttingen
Löffler-Wirth, Henry
Universität Leipzig
Binder, Hans
Universität Leipzig
Arnold, Antje
Fraunhofer-Institut für Zelltherapie und Immunologie IZI  
Stolzing, Alexandra
Fraunhofer-Institut für Zelltherapie und Immunologie IZI  
Journal
Stem cell research  
Open Access
Link
Link
DOI
10.1016/j.scr.2016.03.008
Additional full text version
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Language
English
Fraunhofer-Institut für Zelltherapie und Immunologie IZI  
Keyword(s)
  • induced pluripotent stem cell

  • mRNA

  • reprogramming

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