Fig 1: Characterization of hiPSCs differentiated using two differentiation protocols shows addition of DKK1i effectively maintains FOXL2 expression. (A) Schematic of the two different protocols tested on BM9 cells with the modification of either adding DKK1i alone or adding DKK1i and FGF2. Arrows refer to the timepoints where cells were collected to assess predicted cell types along this differentiation protocol. BMP4 = bone morphogenetic protein 4, DKK1i = DKK1 inhibitor, FST = Follistatin, FGF2 = fibroblast growth factor 2, RA = retinoic acid, SHH = Sonic hedgehog, EGF = epidermal growth factor, IM = intermediate mesoderm, GLC = granulosa-like cells. (B-E) Relative fold change of transcripts POU5F1, DKK1 (B), intermediate mesoderm (IM) markers PAX2, LHX1, and WT1 (C), bipotential gonad markers GATA4 and NR5A1 (D), and granulosa cell marker FOXL2 (E) of BM9 cells differentiated with the two protocols, detected using RT-PCR. All data shown are normalized to day 0 as control within each cell line. Bar graphs show mean of n = 3 experimental replicates ± SEM (each experimental replicate is the mean of 3 technical replicates). Asterisks above bars indicate significance by ordinary one-way ANOVA with Dunnett’s multiple comparisons test, with a single pooled variance, compared to day 0: **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. (F and G) Representative immunofluorescence images of BM9s collected at days 0 and 5 (F) and quantification of the percent of FOXL2-positive cells out of the total nuclei (G) collected during both protocols. See Fig. S1 for representative images of other timepoints. Bar graphs show mean of n = 3 experimental replicates ± SEM (each experimental replicate is the mean of 5––6 technical replicates). DKK1i = DKK1 inhibitor. Scale bars = 100 μm. Any adjustments to the red channel were applied to all images as a flattened layer on Adobe Photoshop. (H and I) Representative blot (H) and quantified protein expression (I) of FOXL2 expression in BM9 cells differentiated using the two protocols. Arrow in (H) indicates FOXL2 band size. Bar graphs show mean of n = 3 experimental replicates ± SEM. Significance was assessed by ordinary one-way ANOVA with Dunnett’s multiple comparisons test, with a single pooled variance, compared to day 0. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)