Fig. 2
Characterization of stage-specific growth kinetics, viability and markers during the first four stages of differentiation. A Cell density during four-stage differentiation (n = 15–16 biological replicates, each biological replicate had 2 technical replicates; one-way ANOVA with Tukey post-test; orange dash line = initial seeding density). B Quantification of stage-specific net growth rate (n = 13–15 biological replicates; one-way ANOVA with Tukey post-test). C Ki67 positive cells using flow cytometry (n = 6 biological replicates; Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post-test). D S4D3 cell yield per input hESC (n = 19 biological replicates). E Viability of cells during four-stage differentiation (n = 12–14 biological replicates; one-way ANOVA with Tukey post-test). F Quantification of stage-specific markers using flow cytometry (n = 9–15 biological replicates). G Gene expression profile of stage-specific markers relative to hESCs (n = 8–16 biological replicates; unpaired t-test) H Illustration of the selective lifting for CDX2low population using crystal violet fixation of adherent cells at the end of S4D3. I Gene expression of CDX2, NKX6.1 and PDX1 in S4D3 lifted cells, S4D3 unlifted cells and S4D4 cell aggregates (n = 9–20 biological replicates; CDX2: two-tailed paired t-test; NKX6.1 and PDX1: Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post-test). J Representative images of S4D3 cells before and after overnight aggregation in the AggreWell™ plate, and aggregate diameter distribution from five independent differentiations, scale bar = 500 µm (n = 244–784 aggregates/group; plot shows individual aggregates with median and interquartile range). K Aggregation efficiency of S4D4 cells (n = 14 biological replicates). L Flow cytometry quantification of NKX6.1 + /PDX1 + positive cells at S4D3 and S4D4 (n = 10–12 biological replicates). All box and whisker plots show individual points with median and interquartile range; bar charts show individual points with mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. S1D3 = stage 1 day 3, S2D3 = stage 2 day 3, S3D2 = stage 3 day 2, S4D3 = stage 4 day 3, S4D4 = stage 4 day 4