FIG. 1.

Reprogramming HFFs using synthetic modified mRNA. (A) Synthetic modified mRNAs are translated into proteins. Cells transfected with mRNA cocktail expressing reprogramming factors Klf4, cMyc, Oct4, LIN28, and Sox2. The 15-h time point was chosen for analysis as it corresponds to the average peak expression time post-transfection of the different reprogramming factors [25]. (B) More than 75% cells express reprogramming factors 15 h after mRNA transfection. The cells were fixed and immunostained for Oct4, Klf4, Sox2, c-Myc, and LIN28 15 h post-transfection with mRNA reprogramming cocktail. Nuclei were visualized with Hoechst 33342. Total cell number was calculated. Data are presented as the mean of cells positive for Oct4, Klf4, Sox2, c-Myc, and LIN28 ± standard error of the mean (n = 3). (C) Daily transfection had a deleterious effect on the cells; vast majority did not survive. Day 0: the cells before the first transfection demonstrate normal morphology and density. Day 3: decreased cell density and cell rounding indicate a high apoptotic rate. Day 6: massive apoptosis is seen. Surviving cells start undergoing mesenchymal epithelial transformation and adopt an epithelial morphology. Day 9: surviving cells continue to transition to an epithelial morphology. Day 12: first signs of compaction and colony formation are noted. Day 15: increased compaction. Cells become smaller and more hESC like. (D) Inhibitory effect of pifithrin-α on p53-mediated transcription. qPCR expression analyses of the two p53 target genes, BAX, and p21 at Day 4 of reprogramming showed decrease in BAX, but not p21 mRNA levels (n = 3 biological replicates). Transfected samples are compared with the negative control (untransfected BJ cells). Data are given by normalized mean ± standard error of the mean (n = 3); statistical significance was calculated by two-way analysis of variance with Tukey post hoc test. ****P < 0.0001. (E) An average reprogramming efficiency from two independent experiments was calculated as number of trypan blue positive colonies per total number of cells plated per well of six-well dish. HFF, human foreskin fibroblast; hESC, human embryonic stem cell; qPCR, quantitative reverse transcriptase–polymerase chain reaction.