Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons

Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar Karuppagounder, Shuli Xia, Valina Dawson, Ted M Dawson, John J Laterra, Jianmin Zhang, Mingyao Ying

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
JournalStem cells translational medicine
DOIs
StateAccepted/In press - Jan 1 2018

Fingerprint

Dopaminergic Neurons
Cell Differentiation
Myosin Type II
Messenger RNA
Induced Pluripotent Stem Cells
Mesencephalon
Neurons
Transcription Factors
Pluripotent Stem Cells
Cell Lineage
Drug Discovery
Cell- and Tissue-Based Therapy
Proteomics
Parkinson Disease
Carrier Proteins
Proteins
Adenosine Triphosphate
Genome
Viruses

Keywords

  • Dopaminergic neuron
  • Induced pluripotent stem cell
  • Motor protein
  • Myosin
  • Parkinson's disease
  • Proneural transcription factor
  • Protein phosphorylation

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

Cite this

Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. / Xue, Yingchao; Zhan, Xiping; Sun, Shisheng; Karuppagounder, Senthilkumar; Xia, Shuli; Dawson, Valina; Dawson, Ted M; Laterra, John J; Zhang, Jianmin; Ying, Mingyao.

In: Stem cells translational medicine, 01.01.2018.

Research output: Contribution to journalArticle

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AU - Xue, Yingchao

AU - Zhan, Xiping

AU - Sun, Shisheng

AU - Karuppagounder, Senthilkumar

AU - Xia, Shuli

AU - Dawson, Valina

AU - Dawson, Ted M

AU - Laterra, John J

AU - Zhang, Jianmin

AU - Ying, Mingyao

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