Fig. 2: Reprogramming fibroblasts into iMPCs with synthetic MyoD-mRNA.

a A schematic depicting the plasmid construct utilized to generate synthetic MyoD-mRNA. Cds, coding sequence; UTR, untranslated region; ARCA, anti-reverse cap analog. b Representative immunofluorescence images of MYOD expression in MEFs transfected with MyoD-mRNA either 5 or 24 h post treatment. Scale bar, 100 μm. Scale bar inlay, 100 μm. c Quantification of MYOD⁺ cells as assessed by immunofluorescence. Data are shown as mean ± SD. N = 3, either 1 or 2 random fields of view were quantified for each cell line. Significance is determined by a mixed-effects analysis using Tukey’s multiple comparison’s test with a single pooled variance, ns=non-significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. d Immunofluorescence staining for the indicated proteins in MEFs transfected 4x with synthetic MyoD-mRNA and stained at day 5. Scale bar, 100 μm. e Timeline of experimental design to produce transgene-free iMPCs. f Top: Images showing ntdTomato⁺ cells following reprogramming of Pax7-CreERT2; R26-LSL-ntdTomato MEFs with MyoD-mRNA+F/R/C at day 15. Cells were labeled with 4-OHT three days prior to analysis. Bottom: FACS-analysis of ntdTomato⁺ cells corresponding to the experiment shown above. Scale bar, 100 μm. g A UMAP projection showing 3517 cells comprising a MyoD-mRNA-derived iMPC clone at passage 5. h Dot plot indicating gene expression level of selected genes in each respective cluster. Average gene expression across all cells in each cluster is shown using a color scale. The percentage of cells expressing the indicated genes is shown as dot size. i A UMAP projection showing the expression level of selected myogenic genes across all cells via a color gradient. j A UMAP projection of single-cell trajectory as indicated by RNA velocity and colored by the latent time of the underlying cellular process.