Fig. 4

Reprogramming factor dosage selected via flow cytometry impacts iPS cell phenotype. a Schematic detailing procedure for isolating colonies reprogrammed with various plasmid dosages. As previously, cells are reprogrammed using our tagged episomal set. Plasmids were also titrated to favor the isolation of OKsm cells (see Methods). Prior to qRT-PCR, clones were passaged eight times to promote stable colonies. Sorting for the pluripotency marker Tra-1-60 and subsequent qRT-PCR for hMEG3 are subsequently performed. b Representative sort and gating strategy for isolating OKsm cells. Here, the ‘sm’ (low blue and green) population was 2.83% of the ‘OK’ (high red and far red) population or 0.11% of the total single events. c Reprogramming efficiency of OKSM vs OKsm sorted cells. N > 3, not significantly different p value. Error bars represent SEM. p = 0.15. d. Expression by qRT-PCR of the imprinted gene hMEG3 at passage 9 post reprogramming within the Tra-1-60+ population of cells (relative expression to GAPDH). Cells reprogrammed with OKsm (red bars) and OKSM (purple) are shown, each different clone designated by a letter or number. Dashed lines represent mean of each group. Clone names reflect the reprogramming method. N = 10 clones, p = 0.041. Error bars represent SEM (Color figure online). OKSM Oct3/4 + shp53, Klf, Sox2, and L-Myc + Lin28