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

Figure 6

Antibiotic treatment causes cellular apoptosis during differentiation of H9 cells towards hepatic and neural fate. (a), (b) The cells (passage number 37) were seeded in 96 well plates for MTT assay upon hepatic and neural differentiation in the presence of 0, 10, 25, 50, and 200 μg/ml gentamicin combined without or with pen-strep (100 U/ml-100 μg/ml). The absorbance was recorded at 560 nM on day 5 and day 10 of hepatic and neural differentiation, respectively. Bars represent absorbance recorded at 560 nm. (c), (d) The release of LDH in the culture media was tested on 50 μl of media from day 5 and day 10 of hepatic and neural differentiation, respectively. As positive control, nontreated cells from the same stage of differentiation were completely lysed to detect maximum LDH release. Bars represent absorbance recorded at 490 nm which is shown here as percentage of the values for untreated (without antibiotic) samples set as 100%. Error bars represent standard deviation. The results are representative of three independent experiments. , , and . (e) For TUNEL assay, cells (passage number 44) were seeded in 96 well plates for hepatic and neural differentiation in the presence of 0, 10, 25, 50, and 200 μg/ml gentamicin combined without or with pen-strep (100 U/ml-100 ug/ml). Images for TUNEL positive cells were captured by light microscope. (f), (g) Quantitation for TUNEL assay was made by counting TUNEL positive cells from each antibiotic treated well (five fields, one in the center and four in each corners of the well). (h), (i) The protein expression levels of cellular apoptotic markers PARP (full length and cleaved) and caspase-3 (pro and cleaved) were analyzed using Western blot for samples treated with antibiotic concentrations as indicated on day 5 and day 10 of hepatic- and neural-differentiated cells, respectively. The expression of GAPDH was used as loading control. The results are representative of three independent experiments. , , and .

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