Figure 4.

Inhibition of Members of the miR-199a/214 Cluster by miRNA Sponges in Differentiating 2D Cultures

MAN13 hESCs transduced either with control (EGFP-only) lentiviral vectors or with vectors expressing “sponge” inhibitors against miR-199a-5p, miR199a-3p, miR-214-5p, or miR-214-3p schematically shown in (A) were differentiated in 2D and immunostained with WT1 or CDH1 antibodies at the end (day 30) of the protocol.

(B–G) Inhibition of miR-199a-3p caused a significant decrease in the extent of the WT1⁺-stained area (MM) within developing cell aggregates (examples outlined) in differentiating cultures. Quantified in (G): mean ± SEM; n = 3 independent differentiation experiments; ^∗p < 0.05, one-sample t test; individual p values are also shown for anti-miR-199a-3p and anti-miR-214-3p). Scale bars, 120 μm.

(H and I) Low-magnification images showing that inhibition of miR-214-3p results in more elongated epithelial structures (CDH1⁺) in the cultures. Scale bars, 600 μm.

(J and K) Examples of individual CDH1⁺ tubules showing normal (control) versus elongated (anti-miR-214-3p) phenotype. Scale bars, 60 μm.

See also Figures S2 and S3.