Rapid Differentiation From Human Induced Pluripotent Stem Cells Into Functional Oligodendrocytes Using Synthetic Modified Olig2 mRNA

Abstract:

Background: Transcription factors (TFs) have been introduced to drive highly efficient differentiation of human induced pluripotent stem cells (hiPSCs) into lineage-specific oligodendrocytes (OLs). However, effective strategies currently rely mainly on genome-integrating viruses. To facilitate the translation of hiPSC-derived OLs into clinical practice, a synthetic modified messenger RNA (smRNA) reprogramming method that generates transgene-free OLs has been developed.

Methods: AnsmRNA encoding Olig2, a key TF in OL development, with a defined phosphorylation site modification serine 147 replaced with alanine, Olig2^S147A, was designed to reprogram hiPSCs into OLs. Proteomics were used to identify Olig2^S147A-binding proteins that positively mediated of Olig2^S147A-driven OL differentiation.

Results: We demonstrated that repeated administration of the smRNA encoding Olig2 ^S147A led to higher and more stable protein expression. Using the single-mutant Olig2 smRNA with morphogens, we established a 6-day smRNA transfection protocol, and glial induction led to rapid NG2⁺ OL progenitor cell (OPC) generation (>70% purity) from hiPSC-derived neural progenitor cells (NPCs). The smRNA-induced NG2⁺ OPCs matured into functional OLs and myelinated nanofibers in vitro. Moreover, when transplanted into mice with cuprizone-induced demyelination, smRNA-induced OPCs promoted remyelination ex vivo. A proteomic analysis of Olig2-binding proteins indicated that the heat shock protein 70 (HSP70) complex bound Olig2. The HSP70 complex bound more strongly to Olig2 with the modified phosphorylation site than to wild type Olig2. VER-155008, an HSP70 complex antagonist, and ML346, an HSP70 complex agonist, inhibited and promoted Olig2 transcriptional activity and efficient OL generation, respectively.Conclusions: We present a very safe and efficient smRNA-driven strategy for hiPSC differentiation into OLs, which might be utilized for disease modeling, drug discovery, and/or therapeutic OPC/OL transplantation in neurodegenerative disease.