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Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons

Proneural transcription factors (TFs) drive highly efficient differentiation of pluripotent stem cells to lineage‐specific neurons. However, current strategies mainly rely on genome‐integrating viruses. Here, we used synthetic mRNAs coding two proneural TFs (Atoh1 and Ngn2) to differentiate induced...

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Autores principales: Xue, Yingchao, Zhan, Xiping, Sun, Shisheng, Karuppagounder, Senthilkumar S., Xia, Shuli, Dawson, Valina L., Dawson, Ted M., Laterra, John, Zhang, Jianmin, Ying, Mingyao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons, Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344911/
https://www.ncbi.nlm.nih.gov/pubmed/30387318
http://dx.doi.org/10.1002/sctm.18-0036
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author Xue, Yingchao
Zhan, Xiping
Sun, Shisheng
Karuppagounder, Senthilkumar S.
Xia, Shuli
Dawson, Valina L.
Dawson, Ted M.
Laterra, John
Zhang, Jianmin
Ying, Mingyao
author_facet Xue, Yingchao
Zhan, Xiping
Sun, Shisheng
Karuppagounder, Senthilkumar S.
Xia, Shuli
Dawson, Valina L.
Dawson, Ted M.
Laterra, John
Zhang, Jianmin
Ying, Mingyao
author_sort Xue, Yingchao
collection PubMed
description Proneural transcription factors (TFs) drive highly efficient differentiation of pluripotent stem cells to lineage‐specific neurons. However, current strategies mainly rely on genome‐integrating viruses. Here, we used synthetic mRNAs coding two proneural TFs (Atoh1 and Ngn2) to differentiate induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (mDA) neurons. mRNAs coding Atoh1 and Ngn2 with defined phosphosite modifications led to higher and more stable protein expression, and induced more efficient neuron conversion, as compared to mRNAs coding wild‐type proteins. Using these two modified mRNAs with morphogens, we established a 5‐day protocol that can rapidly generate mDA neurons with >90% purity from normal and Parkinson's disease iPSCs. After in vitro maturation, these mRNA‐induced mDA (miDA) neurons recapitulate key biochemical and electrophysiological features of primary mDA neurons and can provide high‐content neuron cultures for drug discovery. Proteomic analysis of Atoh1‐binding proteins identified the nonmuscle myosin II (NM‐II) complex as a new binding partner of nuclear Atoh1. The NM‐II complex, commonly known as an ATP‐dependent molecular motor, binds more strongly to phosphosite‐modified Atoh1 than the wild type. Blebbistatin, an NM‐II complex antagonist, and bradykinin, an NM‐II complex agonist, inhibited and promoted, respectively, the transcriptional activity of Atoh1 and the efficiency of miDA neuron generation. These findings established the first mRNA‐driven strategy for efficient iPSC differentiation to mDA neurons. We further identified the NM‐II complex as a positive modulator of Atoh1‐driven neuron differentiation. The methodology described here will facilitate the development of mRNA‐driven differentiation strategies for generating iPSC‐derived progenies widely applicable to disease modeling and cell replacement therapy. stem cells translational medicine 2019;8:112&12
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spelling pubmed-63449112019-01-28 Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons Xue, Yingchao Zhan, Xiping Sun, Shisheng Karuppagounder, Senthilkumar S. Xia, Shuli Dawson, Valina L. Dawson, Ted M. Laterra, John Zhang, Jianmin Ying, Mingyao Stem Cells Transl Med Pluripotent Stem Cells Proneural transcription factors (TFs) drive highly efficient differentiation of pluripotent stem cells to lineage‐specific neurons. However, current strategies mainly rely on genome‐integrating viruses. Here, we used synthetic mRNAs coding two proneural TFs (Atoh1 and Ngn2) to differentiate induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (mDA) neurons. mRNAs coding Atoh1 and Ngn2 with defined phosphosite modifications led to higher and more stable protein expression, and induced more efficient neuron conversion, as compared to mRNAs coding wild‐type proteins. Using these two modified mRNAs with morphogens, we established a 5‐day protocol that can rapidly generate mDA neurons with >90% purity from normal and Parkinson's disease iPSCs. After in vitro maturation, these mRNA‐induced mDA (miDA) neurons recapitulate key biochemical and electrophysiological features of primary mDA neurons and can provide high‐content neuron cultures for drug discovery. Proteomic analysis of Atoh1‐binding proteins identified the nonmuscle myosin II (NM‐II) complex as a new binding partner of nuclear Atoh1. The NM‐II complex, commonly known as an ATP‐dependent molecular motor, binds more strongly to phosphosite‐modified Atoh1 than the wild type. Blebbistatin, an NM‐II complex antagonist, and bradykinin, an NM‐II complex agonist, inhibited and promoted, respectively, the transcriptional activity of Atoh1 and the efficiency of miDA neuron generation. These findings established the first mRNA‐driven strategy for efficient iPSC differentiation to mDA neurons. We further identified the NM‐II complex as a positive modulator of Atoh1‐driven neuron differentiation. The methodology described here will facilitate the development of mRNA‐driven differentiation strategies for generating iPSC‐derived progenies widely applicable to disease modeling and cell replacement therapy. stem cells translational medicine 2019;8:112&12 John Wiley & Sons, Inc. 2018-11-01 /pmc/articles/PMC6344911/ /pubmed/30387318 http://dx.doi.org/10.1002/sctm.18-0036 Text en © 2018 The Authors stem cells translational medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Pluripotent Stem Cells
Xue, Yingchao
Zhan, Xiping
Sun, Shisheng
Karuppagounder, Senthilkumar S.
Xia, Shuli
Dawson, Valina L.
Dawson, Ted M.
Laterra, John
Zhang, Jianmin
Ying, Mingyao
Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons
title Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons
title_full Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons
title_fullStr Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons
title_full_unstemmed Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons
title_short Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons
title_sort synthetic mrnas drive highly efficient ips cell differentiation to dopaminergic neurons
topic Pluripotent Stem Cells
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344911/
https://www.ncbi.nlm.nih.gov/pubmed/30387318
http://dx.doi.org/10.1002/sctm.18-0036
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