Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons

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. stem cells translational medicine 2019;8:112&12

Transcription Factor-Based Fate Specification and Forward Programming for Neural Regeneration

“…First hints as of why bHLH TFs might be able to orchestrate neuronal fate acquisition were obtained from NPC-to-neuron differentiation paradigms: Ngn1, for instance, specifically binds to E-box motifs at neuronal genes in rat NPCs, acting as a direct transcriptional activator (Sun et al, 2001). In human PSCs, the TFs NGN1, NGN2 and NGN3 seem to even cross-activate each other and induce common proneural down-stream targets including other bHLH TFs such as NEUROD1, NEUROD2 and NEUROD4 (Busskamp et al, 2014;Goparaju et al, 2017;Xue et al, 2019). Such a synergism might not be restricted to the group of NGNs, since the bHLH TF ATOH1 has been shown to induce both, NGN2 and NEUROD1 in human PSCs, and was thus used for forward programming of human PSCs into neurons (Sagal et al, 2014;Xue et al, 2019).…”

“…In human PSCs, the TFs NGN1, NGN2 and NGN3 seem to even cross-activate each other and induce common proneural down-stream targets including other bHLH TFs such as NEUROD1, NEUROD2 and NEUROD4 (Busskamp et al, 2014;Goparaju et al, 2017;Xue et al, 2019). Such a synergism might not be restricted to the group of NGNs, since the bHLH TF ATOH1 has been shown to induce both, NGN2 and NEUROD1 in human PSCs, and was thus used for forward programming of human PSCs into neurons (Sagal et al, 2014;Xue et al, 2019). These observations might indicate that one common mechanism underlying neuronal forward programming of PSCs with bHLH TFs is the activation of a whole network of crossregulated bHLH TFs, including the induction of more downstream Neurod TFs.…”

“…Characterization of the growth factor-treated, ATOH1-overexpressing neuronal cultures on a functional level demonstrated that these neurons possess electrophysiological properties similar to primary rat midbrain dopaminergic neurons and exhibit dopamine release after electrical stimulation at day 36 of differentiation, suggesting actual functionality (Sagal et al, 2014). Very recently, this approach was further improved by establishing a protocol based on combined ATOH1 and NGN2 overexpression in human iPSCs via repetitive mRNA transfections (Xue et al, 2019).…”

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“…As an alternative approach, researchers led by Mingyao Ying (Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, MD) and Jianmin Zhang (Peking Union Medical College, Beijing, China) assessed the ability of synthetic mRNAs coding for proneural TFs (Atoh1 and Ngn2) to differentiate iPSCs into mDA neurons. In their Stem Cells Translational Medicine article , Xue et al report that synthetic mRNAs with defined phosphosite modifications permitted the generation of mDA neurons at >90% purity from normal‐ and PD‐iPSCs following a five‐day protocol. mRNA‐induced mDA neurons displayed the essential biochemical and electrophysiological features of primary mDA neurons following in vitro maturation, thereby highlighting the potential of this mRNA‐based strategy to provide the high numbers of functional cells required for modeling, screening, and cell replacement therapies.…”

“…Human neural stem cells (hNSCs) also represent an exciting cell therapeutic prospect, with the hope that transplanted cells can either differentiate in vivo to replace lost host cells or provide support for endogenous repair mechanisms. In our first Featured Article from Stem Cells Translational Medicine , Xue et al describe the rapid differentiation of both normal‐ and PD‐iPSCs into functional mDA neurons via the application of synthetic mRNAs coding for two proneural transcription factors (TFs) . In a Related Article from Stem Cells , Zuo et al demonstrate how the transplantation of hNSCs into the striatum of PD model mice rescues the unexpected deficits observed in the subventricular zone (SVZ) by eliciting an endogenous regenerative response .…”