Fig. 1.

Reprogramming of somatic cells with adenovirus containing reprogramming factors (RFs) in a single cassette. (A) Schematic representations of adenovirus-GFP (Ad-GFP) (i), and adenoviral vector containing RFs (Ad-SOcMK) (ii). RFs SOX2, OCT3/4, c-MYC and KLF4 are controlled individually by separate CMV promoters in a single cassette. (B) Transduction of human IMR90 cells with Ad-SOcMK, but not with Ad-GFP, results in the appearance of cell clumps by days 2-3. Photomicrographs of Ad-GFP- and Ad-SOcMK-transduced cells with GFP expression (upper panel) on day 3 post-transduction. Lower panel: alkaline phosphatase (ALP) cytochemistry assay on day 3. IMR90 cells transduced with Ad-SOcMK stain for ALP on day 3, in contrast to IMR90 cells transduced with Ad-GFP. Representative micrographs of three independent experiments are shown. All images at 10× magnification. (C) IMR90 cells transduced with Ad-SOcMK express exogenous RFs. Protein extracts from the harvested IMR90 cells transduced with Ad-GFP or Ad-SOcMK on day 3 were subjected to western blot analyses. The blots were re-probed for actin as an internal loading control. The blot represents one of three independent experiments. (D) Switch in transcript (upper panel) and protein expression (lower panel) of two key genes involved in the mesenchymal to epithelial transition (MET). THY1, but not CDH1 is expressed in IMR90 cells. On day 3 post-transduction with Ad-SOcMK, strong expression of CDH1 is seen, whereas THY1 expression is suppressed. For RT-PCR, GAPDH is used as internal control, for western blot analysis, actin serves as loading control. (E) Quantification of reprogramming efficiency of IMR90 cells. Synthesized cDNAs were subjected to qPCR analysis to measure the expression levels of THY1 mRNA in Ad-SOcMK-transduced cells at day 3, with GAPDH mRNA as an internal control. Error bars (±) represent standard deviation (n=3). P-values were determined by paired Student's t-test.