Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells

Figure 5

Effects of the chemical mixture (DEP, Hg, cotinine, Se, and S-421) on cellular differentiation. (a) Effects of the timing of chemical exposure on EB formation. EBs derived from hiPSCs were cultured using three culture conditions: I, solvent-treated control; II and III, cells treated with the chemical mixture for 4 days before differentiation. EB formation was then induced for up to 24 days in the absence (II) or presence (III) of the chemical mixture (left panel). The right panel shows images of EBs on day 24. Scale bar = 250 μm. −: solvent only; +: exposure to serum concentrations of the chemical mixture. Experiments were performed thrice independently. (b) Effects of the five chemicals on neural differentiation. The culture conditions used were the same as in (a). Differentiated cells were analyzed on day 20 (right panel). Enlarged images are shown as “a” and “b” for conditions II and III, respectively. Scale bar = 200 μm. Experiments were performed twice independently. (c) After 24 days of neural differentiation in culture conditions I and II, cells were stained with antibodies for the neural marker βIII-tubulin, and the βIII-tubulin-positive area (%) in 150 images was measured using ImageJ software. The data are presented as means ± SE. (d) Neural marker gene expression. On day 24, cells grown in culture conditions I and II were harvested, and the expression levels of the neural marker genes NES, MAP2, and PAX6 were assessed using RT-PCR. The relative expression levels were normalized to that of GAPDH. The expression levels are shown as mean ± SD (). Statistical comparisons of the expression level were performed using Student’s t-test. The -value of NES, MAP2, and PAX6 was 0.081, 0.015, and 0.065, respectively. . (e) Summary of cellular differentiation in chemical-exposed hiPSCs. −: solvent only; +: exposure to serum concentrations of DEP, Hg, cotinine, Se, and S-421.

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