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. 2015 Feb 4;6:6188. doi: 10.1038/ncomms7188

Figure 1. AA combined with 2i enhances reprogramming efficiency.

Figure 1

(a) Counts of Nanog-positive colonies on day 17 of MEF reprogramming. Error bars represent standard deviation of four replicates. AA, ascorbic acid; 2i, MAP kinase inhibitor (PD-0325901)+glycogen synthetase kinase inhibitor (CHIR-99021). (b) Same as above but for adult fibroblasts. Technical replicates are stacked. Rep1 and Rep2 indicate two different genetic backgrounds. (c) Inset—Scheme of experiment: JSS MEFs received doxycycline (dox) for OSKM induction from day 0 to day 17 and were treated with DMSO, AA, 2i or AA+2i for the days indicated (grey bars). Counts of Nanog-positive colonies for four replicates. *P<0.05 **P<0.01 assessed by t-test. (d) Top panel—Scheme of experiment: Cells were sorted for SSEA1 and lack of Thy1 expression (Thy1−SSEA1+) on day 9 after dox induction. Sorted cells were exposed to DMSO, AA, 2i or AA+2i until day 13 or 16 post dox induction. Bottom panel—Bar graphs show combined results from two biological replicates (A and B) for Nanog-positive colonies after 4 (left) or 7 (right) days of treatment. *P<0.05 by t-test. (e) Left panel—Bar graph representing GFP reporter expression (under the control of the endogenous Nanog promoter) in pre-iPSCs upon exposure to AA and/or 2i as compared with DMSO by flow cytometry. Right panel—Counts of Nanog-GFP-positive ESC-like colonies that represent the Nanog-GFP-positive cells in the left panel. Inset: Image of Nanog-GFP-positive colonies upon exposure to AA+2i. Scale bar, 250 μm. Error bars represent standard deviation of three biological replicates. Asterisk indicates significance *P<0.05, ****P<0.0001 assessed by t-test. (f) Quantitative reverse transcription—PCR of various differentiation markers on day 7 post in vitro differentiation of two independent iPSC lines #1 and #2 derived from pre-iPSCs after AA+2i treatment. Dotted line represents levels on day 0 which was set to 1. (g) Images depicting the three germ layers from teratomas formed by two independent AA+2i-derived iPSC clones. Scale bar, 100 μm.