FIGURE 3.

Engineered cardiopoietic stem cells (eCP) demonstrate elevated antioxidant capacity. A, Oxidative stress was induced in AC16 human cardiomyocytes using the free‐radical generator LY83583. Four treatment groups included; culture medium alone (Media), plus OptiMEM (+OptiMEM), plus adipose‐derived mesenchymal stem cell‐derived conditioned media (+AMSC CM), or plus Brachyury transfected engineered cardiopoietic stem cell‐derived conditioned media (+eCP CM). In each treatment group, 20 μL of conditioned media was added to 200 μL of regular media per well. Caspase 3/7 Green and NucLight Red identified apoptotic cells and cell nuclei, respectively (top panels). Significant decrease in apoptotic cardiomyocytes (bottom panels) indicated a protective effect of eCP conditioned media. Numbers of biological replicates (n) were nine per group at each time point. Scale bar = 100 μm. Antioxidant capacity was consistently detected as measured by: expression of antioxidant proteins SOD2 and SOD3 in total cell lysate (Western blot, n = 3; B); superoxide dismutase (SOD) activity in conditioned media (n = 3; C); and total antioxidant capacity in conditioned media (n = 3; D). OptiMEM served as control. *P < .05; **P < .01; ***P < .001 with one‐way ANOVA followed by a post hoc Bonferroni test (A, C, D) or Student's t test (B). ANOVA, analysis of variance