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

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-Methyltrans...

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Main Authors:	Leili Rohani, Claire Fabian, H. Holland, Yahaira Naaldijk, R. Dressel, H. Loffler-Wirth, Hans Binder, Antje Arnold, Alexandra Stolzing
Format:	Default Article
Published:	2016
Subjects:	Mechanical engineering not elsewhere classified Induced pluripotent stem cell Reprogramming mRNA Mechanical Engineering not elsewhere classified
Online Access:	https://hdl.handle.net/2134/20950
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id	rr-article-9563450
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spelling	rr-article-95634502016-01-01T00:00:00Z Generation of human induced pluripotent stem cells using non-synthetic mRNA. Leili Rohani (7212278) Claire Fabian (3933104) H. Holland (7212281) Yahaira Naaldijk (3933095) R. Dressel (7212284) H. Loffler-Wirth (7212287) Hans Binder (128524) Antje Arnold (7212290) Alexandra Stolzing (1251348) Mechanical engineering not elsewhere classified Induced pluripotent stem cell Reprogramming mRNA Mechanical Engineering not elsewhere classified 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. 2016-01-01T00:00:00Z Text Journal contribution 2134/20950 https://figshare.com/articles/journal_contribution/Generation_of_human_induced_pluripotent_stem_cells_using_non-synthetic_mRNA_/9563450 CC BY-NC-ND 4.0
institution	Loughborough University
collection	Figshare
topic	Mechanical engineering not elsewhere classified Induced pluripotent stem cell Reprogramming mRNA Mechanical Engineering not elsewhere classified
spellingShingle	Mechanical engineering not elsewhere classified Induced pluripotent stem cell Reprogramming mRNA Mechanical Engineering not elsewhere classified Leili Rohani Claire Fabian H. Holland Yahaira Naaldijk R. Dressel H. Loffler-Wirth Hans Binder Antje Arnold Alexandra Stolzing Generation of human induced pluripotent stem cells using non-synthetic mRNA.
description	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.
format	Default Article
author	Leili Rohani Claire Fabian H. Holland Yahaira Naaldijk R. Dressel H. Loffler-Wirth Hans Binder Antje Arnold Alexandra Stolzing
author_facet	Leili Rohani Claire Fabian H. Holland Yahaira Naaldijk R. Dressel H. Loffler-Wirth Hans Binder Antje Arnold Alexandra Stolzing
author_sort	Leili Rohani (7212278)
title	Generation of human induced pluripotent stem cells using non-synthetic mRNA.
title_short	Generation of human induced pluripotent stem cells using non-synthetic mRNA.
title_full	Generation of human induced pluripotent stem cells using non-synthetic mRNA.
title_fullStr	Generation of human induced pluripotent stem cells using non-synthetic mRNA.
title_full_unstemmed	Generation of human induced pluripotent stem cells using non-synthetic mRNA.
title_sort	generation of human induced pluripotent stem cells using non-synthetic mrna.
publishDate	2016
url	https://hdl.handle.net/2134/20950
_version_	1797374709636005888

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

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