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In order to realize the practical use of human pluripotent stem cell (hPSC)-derived cardiomyocytes for the purpose of clinical use or cardiovascular research, the generation of large numbers of highly purified cardiomyocytes should be achieved. Here, we show an efficient method for cardiac differentiation of human induced pluripotent stem cells (hiPSCs) in chemically defined conditions and purification of hiPSC-derived cardiomyocytes using a reporter system. Regulation of the Wnt/β-catenin signaling pathway is implicated in the induction of the cardiac differentiation of hPSCs. We increased cardiac differentiation efficiency of hiPSCs in chemically defined conditions through combined treatment with XAV939, a tankyrase inhibitor and IWP2, a porcupine inhibitor and optimized concentrations. Although cardiac differentiation efficiency was high (>80%), it was difficult to suppress differentiation into non-cardiac cells, Therefore, we applied a lentiviral reporter system, wherein green fluorescence protein (GFP) and Zeocin-resistant gene are driven by promoter activation of a gene (TNNT2) encoding cardiac troponin T (cTnT), a cardiac-specific protein, to exclude non-cardiomyocytes from differentiated cell populations. We transduced this reporter construct into differentiated cells using a lentiviral vector and then obtained highly purified hiPSC-derived cardiomyocytes by treatment with the lowest effective dose of Zeocin. We significantly increased transgenic efficiency through manipulation of the cells in which the differentiated cells were simultaneously infected with virus and re-plated after single-cell dissociation. Purified cells specifically expressed GFP, cTnT, displayed typical properties of cardiomyocytes. This study provides an efficient strategy for obtaining large quantities of highly purified hPSC-derived cardiomyocytes for application in regenerative medicine and biomedical research.

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation.

Abstract. The present study examined the role of human cytochrome P450 2J2 (CYP2J2) on cell proliferation and resistance to an anticancer agent using stable hepatocellular carcinoma HepG2 cells overexpressing CYP2J2. Overexpression of CYP2J2 significantly increased HepG2 cell proliferation and the expression levels of cell cycle regulatory proteins, including cyclin D1, cyclin E, cyclin-dependent kinase (Cdk)2 and Cdk4. CYP2J2-overexpressing HepG2 cells exhibited high levels of Akt phosphorylation compared with those observed in wild-type HepG2 cells. Although Akt phosphorylation in both cell lines was significantly attenuated by LY294002, a specific phosphoinositide 3-kinase/Akt signaling inhibitor, the levels of Akt phosphorylation following treatment with LY294002 were higher in CYP2J2-overexpressing HepG2 cells than in wild-type HepG2 cells. Cell counting revealed that proliferation was reduced by LY294002 in both cell lines; however, CYP2J2-overexpressing HepG2 cell numbers were higher than those of wild-type HepG2 cells following treatment with LY294002. These results indicated that increased cell proliferation by CYP2J2 overexpression is mediated by increased Akt activity. It was also demonstrated that doxorubicin, an anticancer agent, reduced cell viability, induced a significant increase in the B-cell lymphoma (Bcl)-2 associated X protein (Bax)/Bcl-2 ratio and decreased pro-caspase-3 levels in wild-type HepG2 cells. However, the doxorubicin-induced reduction in cell viability was significantly attenuated by enhanced upregulation of CYP2J2 expression. The increase in the Bax/Bcl-2 ratio and the decrease in pro-caspase-3 levels were also recovered by CYP2J2 overexpression. In conclusion, CYP2J2 serves important roles in cancer cell proliferation and resistance to the anticancer agent doxorubicin in HepG2 cells.