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

Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\'' 2′-O-Methyltransferase enzymecreates 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-γ) or alpha (hIFN-α) 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.

Identiferoai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:14796
Date January 2016
CreatorsRohani, Leili, Fabian, Claire, Holland, Heidrun, Naaldijk, Yahaira, Dressel, Ralf, Löffler-Wirth, Henry, Binder, Hans, Arnold, A., Stolzing, Alexandra
ContributorsFraunhofer-Institut für Zelltherapie und Immunologie, Universität Leipzig, Universität Göttingen, Loughborough University
PublisherElsevier
Source SetsHochschulschriftenserver (HSSS) der SLUB Dresden
LanguageEnglish
Detected LanguageEnglish
Typedoc-type:article, info:eu-repo/semantics/article, doc-type:Text
SourceStem cell research 16(2016) 662-672 doi: 10.1016/j.scr.2016.03.008
Rightsinfo:eu-repo/semantics/openAccess

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