Fig. 1

mRNAs coding phosphosite-modified Olig2 and Ngn2 induce efficient conversion of iPSCs to the MN lineage. (A) Diagram of mRNAs coding phosphosite-modified Ngn2 (N-SA), wild-type Olig2 (O-WT) and phosphosite-modified Olig2 (O-SA). Arrows indicate serine-to-alanine modifications (Ngn2: at 24, 193, 207, 209, 219, 232, 239 and 242 amino acids; Olig2: at 10, 13, 14 amino acids). (B) Control iPSCs (iPSC1) received a single transfection of O-WT or O-SA mRNAs in combination with N-SA mRNAs. Total cellular proteins were harvested at 24 and 48 h for Olig2 western blotting. Protein fold expression normalized to Actin are shown below each lane (O-WT samples = 1.0). (C and D) Schematic diagram of six conditions tested for identifying the most efficient strategy for MN lineage conversion. In all conditions, iPSC1 cells received three daily transfections of N-SA mRNAs in combination with O-WT, O-SA mRNAs and/or SHH/DAPT. Cells at day 4 of differentiation were subjected to qPCR to compare two MN lineage markers (HB9 and Islet1) and the pan-neuronal marker NeuroD1. Gene expression was normalized to the levels of undifferentiated iPSCs. Data represents Mean ± SEM from qPCR with 3 technical replicates, p values are calculated by one-way ANOVA with Tukey’s HSD pos-hoc test