Generation of human induced pluripotent stem cells using non-synthetic mRNA.
journal contributionposted on 2016-04-18, 14:01 authored by Leili Rohani, Claire Fabian, H. Holland, Yahaira Naaldijk, R. Dressel, H. Loffler-Wirth, Hans Binder, Antje Arnold, Alexandra StolzingAlexandra Stolzing
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-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, 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.
The work was supported by funding from the German Federal Ministry of Education and Research (BMBF 1315883).
- Mechanical, Electrical and Manufacturing Engineering
Published inStem cell research
Pages662 - 672
CitationROHANI, L. ...et al., 2016. Generation of human induced pluripotent stem cells using non-synthetic mRNA.. Stem cell research, 16(3), pp. 662-672.
Publisher© The Authors. Published by Elsevier
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Publisher statementThis work is made available according to the conditions of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) licence. Full details of this licence are available at: https://creativecommons.org/licenses/by-nc-nd/4.0/
NotesThis is an Open Access Article. It is published by Elsevier under the Creative Commons Attribution 4.0 Unported Licence (CC BY-NC-ND). Full details of this licence are available at: http://creativecommons.org/licenses/by-nc-nd/4.0/