Figure 4.

Functional Analysis of Mesoderm-Specific miRNAs

(A) Schematic overview of the experimental setup.

(B) Representative flow-cytometric dot plot diagrams after 4 days of ME differentiation (HES3). Transfected miRNA inhibitors or mimics are marked. Indicated numbers represent the percentages of double-positive cells (upper panel: NCAM⁺/PDGFRA⁺; lower panel: NCAM⁺/KDR⁺).

(C and D) Quantification of NCAM⁺/PDGFRA⁺ and NCAM⁺/KDR⁺ cells by flow cytometry at day 4 using the HES3 (C) or HUES8 (D) cell line. Transfection with the indicated mimics/inhibitors was performed at day 2. Values are mean ± SEM, n = 4–8. ANOVA plus Dunnett’s post hoc test compared with the respective NC (C) or Student's t test (D), ^∗p < 0.05 and ^∗∗p < 0.01; n.s., not significant.

(E) Normalized gene expressions of ME markers (PDGFRA, KDR, CD34) at day 4 (HES3). Values are means ± SEM, n = 4. Student's t test, ^∗p < 0.05.

(F) Depicted is a PDGFRA and CD34 staining of the mesodermal NCAM⁺ population at day 4 after randomized or ME differentiation (HES3). Indicated percentages represent the numbers of cells within the quadrant of this particular experiment.

(G) Normalized miRNA expression in the three purified populations of mesodermal NCAM⁺ cells scaled to undifferentiated hESCs (HES3). Values are mean ± SEM, n = 3–6. ANOVA plus Bonferroni's post hoc test, ^∗p < 0.05 and ^∗∗p < 0.01 compared with P⁻ cells. P⁻, NCAM⁺/PDGFRA⁻/CD34⁻ cells; P⁺, NCAM⁺/PDGFRA⁺/CD34⁻ cells; CD34⁺, NCAM⁺/CD34⁺/PDGFRA⁻ cells.

See also Figure S4E.