Protocols for Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons
This collection is a draft, published without a DOI.
Yingchao Xue1,2,
Xiping Zhan3,
Shisheng Sun4,
Senthilkumar S. Karuppagounder5,6,7,
Shuli Xia2,5,
Valina L Dawson5,6,7,8,9,
Ted M Dawson5,6,7,8,10,
John Laterra2,5,8,11,
Jianmin Zhang1,
Mingyao Ying2,5
1Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology;
2Hugo W. Moser Research Institute at Kennedy Krieger;
3Department of Physiology and Biophysics, Howard University;
4College of Life Sciences, Northwest University;
5Department of Neurology, Johns Hopkins University School of Medicine;
6Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine;
7Adrienne Helis Malvin Medical Research Foundation;
8Department of Neuroscience, Johns Hopkins University School of Medicine;
9Department of Physiology, Johns Hopkins University School of Medicine;
10Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine;
11Department of Oncology, Johns Hopkins University School of Medicine
Collection Citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. Protocols for Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. protocols.io https://protocols.io/view/protocols-for-synthetic-mrnas-drive-highly-efficie-9e5h3g6
Manuscript citation:
Xue Y, Zhan X, Sun S, et al. Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Stem Cells Transl Med. 2019;8(2):112–123. doi:10.1002/sctm.18-0036
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
These protocols were published in:
Xue Y, Zhan X, Sun S, et al. Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Stem Cells Transl Med. 2019;8(2):112–123. doi:10.1002/sctm.18-0036
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.
All results reported should represent at least three independent replications. Perform statistical analysis using Prism software. Post hoc tests should include the Student’s t test and the Tukey multiple comparison tests as appropriate. For neurophysiological recordings, the recorded data should first be visualized with Clampfit 9.2 and exported to MATLAB for further analysis and plotting. Visualize recording traces with Igor Pro 6.0. All data to be represented as mean ± SEM.
Safety warnings
Please refer to the Safety Data Sheets (SDS) for safety and environmental hazards.
Before start
Obtain approval to work with human stem cells from an appropriate Institutional Review Board.
