TY - JOUR
T1 - Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons
AU - Xue, Yingchao
AU - Zhan, Xiping
AU - Sun, Shisheng
AU - Karuppagounder, Senthilkumar
AU - Xia, Shuli
AU - Dawson, Valina L.
AU - Dawson, Ted M.
AU - Laterra, John
AU - Zhang, Jianmin
AU - Ying, Mingyao
N1 - Funding Information:
We thank Dr. Joel Jessee from MTI-GlobalStem, Inc. for providing mRNA transfection reagents. This work was supported by the Maryland Stem Cell Research Fund (M.Y. and T.M.D.). This work was supported by the CAMS Initiative for Innovative Medicine (2016-I2M-1-008), the National Key Research and Development Program of China (2016YFA0101001, 2016YFC0903900), and CAMS Central Public Welfare Scientific Research Institute Basal Research Expenses (2017PT31004). The authors acknowledge the Adrienne Helis Malvin Medical Research Foundation and its direct engagement in the continuous active conduct of medical research in conjunction with the Johns Hopkins Hospital and the Johns Hopkins University School of Medicine and the Foundation’s Parkinson’s Disease Program M2015. T.M.D. is the Leonard and Madlyn Abramson Professor in Neurodegenerative Diseases.
Funding Information:
We thank Dr. Joel Jessee from MTI-GlobalStem, Inc. for providing mRNA transfection reagents. This work was supported by the Maryland Stem Cell Research Fund (M.Y. and T.M.D.). This work was supported by the CAMS Initiative for Innovative Medicine (2016-I2M-1-008), the National Key Research and Development Program of China (2016YFA0101001, 2016YFC0903900), and CAMS Central Public Welfare Scientific Research Institute Basal Research Expenses (2017PT31004). The authors acknowledge the Adrienne Helis Malvin Medical Research Foundation and its direct engagement in the continuous active conduct of medical research in conjunction with the Johns Hopkins Hospital and the Johns Hopkins University School of Medicine and the Foundation's Parkinson's Disease Program M2015. T.M.D. is the Leonard and Madlyn Abramson Professor in Neurodegenerative Diseases.
Publisher Copyright:
© 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press
PY - 2019/2
Y1 - 2019/2
N2 - 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.
AB - 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.
KW - Dopaminergic neuron
KW - Induced pluripotent stem cell
KW - Motor protein
KW - Myosin
KW - Parkinson's disease
KW - Proneural transcription factor
KW - Protein phosphorylation
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U2 - 10.1002/sctm.18-0036
DO - 10.1002/sctm.18-0036
M3 - Article
C2 - 30387318
AN - SCOPUS:85056121117
SN - 2157-6564
VL - 8
SP - 112
EP - 123
JO - Stem Cells Translational Medicine
JF - Stem Cells Translational Medicine
IS - 2
ER -