Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

Figure 1

Treatment of H9 cells with antibiotics does not affect their viability, morphology, and the expression of pluripotency markers. (a) MTT assay was performed on H9 cells (passage number 40) grown in 96 well plates, at day 2 or day 5, posttreatments. Bars represent absorbance recorded at 560 nM. (b) Cells (passage number 40) were grown in feeder-free condition on Matrigel-coated plates for 5 days treated with antibiotics as indicated. The cells were fixed and photographed using inverted microscope in the bright field to compare colony morphology. The cells were then stained using anti-Oct4 antibody and imaged with fluorescence microscope. DAPI represents nuclear staining. Scale bar = 100 μm. (c) The Oct4-stained cells were quantified using FACS. The bars represent percentage of cells positive for Oct4 in comparison to no antibody control. (d) The expression of RNA on cells (passage number 40) treated with antibiotic concentrations as indicated on day 5 was analyzed by qRT-PCR using primers specific for Oct4, Sox2, and Nanog genes. The expression of Polr2g gene was used as normalizer. The bars represent normalized mRNA expression with values of untreated samples set as 1. (e) The protein levels of Oct4, Sox2, and Nanog were analyzed as indicated. The expression of GAPDH was used as loading control. Each experiment was performed at least three times. Values are expressed as mean ± SD in each group.

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