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

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posted on 18.04.2016 by Leili Rohani, Claire Fabian, H. Holland, Yahaira Naaldijk, R. Dressel, H. Loffler-Wirth, Hans Binder, Antje Arnold, Alexandra 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.

Funding

The work was supported by funding from the German Federal Ministry of Education and Research (BMBF 1315883).

History

School

  • Mechanical, Electrical and Manufacturing Engineering

Published in

Stem cell research

Volume

16

Issue

3

Pages

662 - 672

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.

Publisher

© The Authors. Published by Elsevier

Version

VoR (Version of Record)

Publisher statement

This 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/

Publication date

2016

Notes

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/

ISSN

1873-5061

eISSN

1876-7753

Language

en

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