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Title: Generation of human induced pluripotent stem cells using non-synthetic mRNA.
Authors: Rohani, Leili
Fabian, Claire
Holland, H.
Naaldijk, Yahaira
Dressel, R.
Loffler-Wirth, H.
Binder, Hans
Arnold, Antje
Stolzing, Alexandra
Keywords: Induced pluripotent stem cell
Issue Date: 2016
Publisher: © The Authors. Published by Elsevier
Citation: ROHANI, L. ...et al., 2016. Generation of human induced pluripotent stem cells using non-synthetic mRNA.. Stem cell research, 16(3), pp. 662-672.
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-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.
Description: This 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/
Sponsor: The work was supported by funding from the German Federal Ministry of Education and Research (BMBF 1315883).
Version: Published
DOI: 10.1016/j.scr.2016.03.008
URI: https://dspace.lboro.ac.uk/2134/20950
Publisher Link: http://dx.doi.org/10.1016/j.scr.2016.03.008
ISSN: 1873-5061
Appears in Collections:Published Articles (Mechanical, Electrical and Manufacturing Engineering)

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