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-Methyltransferas...
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doaj-82cc7d3262784112b047ed98bebe329c2020-11-24T20:56:01ZengElsevierStem Cell Research1873-50611876-77532016-05-0116366267210.1016/j.scr.2016.03.008Generation of human induced pluripotent stem cells using non-synthetic mRNAL. Rohani0C. Fabian1H. Holland2Y. Naaldijk3R. Dressel4H. Löffler-Wirth5H. Binder6A. Arnold7A. Stolzing8Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, GermanyFraunhofer Institute for Cell Therapy and Immunology, Leipzig, GermanyTranslational Centre for Regenerative Medicine (TRM), University Leipzig, GermanyFraunhofer Institute for Cell Therapy and Immunology, Leipzig, GermanyInstitute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, GermanyInterdisciplinary Centre for Bioinformatics (IZBI), Leipzig University, Leipzig, GermanyInterdisciplinary Centre for Bioinformatics (IZBI), Leipzig University, Leipzig, GermanyFraunhofer Institute for Cell Therapy and Immunology, Leipzig, GermanyFraunhofer Institute for Cell Therapy and Immunology, Leipzig, GermanyHere 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 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.http://www.sciencedirect.com/science/article/pii/S187350611630006XInduced pluripotent stem cellReprogrammingmRNA |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
L. Rohani C. Fabian H. Holland Y. Naaldijk R. Dressel H. Löffler-Wirth H. Binder A. Arnold A. Stolzing |
spellingShingle |
L. Rohani C. Fabian H. Holland Y. Naaldijk R. Dressel H. Löffler-Wirth H. Binder A. Arnold A. Stolzing Generation of human induced pluripotent stem cells using non-synthetic mRNA Stem Cell Research Induced pluripotent stem cell Reprogramming mRNA |
author_facet |
L. Rohani C. Fabian H. Holland Y. Naaldijk R. Dressel H. Löffler-Wirth H. Binder A. Arnold A. Stolzing |
author_sort |
L. Rohani |
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 |
publisher |
Elsevier |
series |
Stem Cell Research |
issn |
1873-5061 1876-7753 |
publishDate |
2016-05-01 |
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 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. |
topic |
Induced pluripotent stem cell Reprogramming mRNA |
url |
http://www.sciencedirect.com/science/article/pii/S187350611630006X |
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