Inducible effects of icariin, icaritin, and desmethylicaritin on directional differentiation of embryonic stem cells into cardiomyocytes in vitro

Aim: To investigate the possible inducible effects of icariin, icaritin, and desmethylicaritin on the directional differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro. Methods: ES cells were cultivated as embryoid bodies (EBs) in hanging drops with icariin, icaritin, or desmethylicaritin. ES cells treated with retinoic acid and with solvent were used as positive and negative respectively. The cardiomyocytes derived from the ES cells were verified, using immunocytochemistry. The expression of cardiac developmental-dependent genes was detected using the reverse transcription-polymerase chain reaction (RT-PCR) method. Cell cycle distribution and apoptosis were analyzed using flow cytometry to determine the partly inducible effect mechanisms involved. Results: The total percentage of beating EBs treated with 1 x 10(-7) mol/L icariin, icaritin, ordesmethylicaritin was 87% (P < 0.01), 59% (P < 0.01), and 49%, respectively. All the beating cardiomyocytes derived from the ES cells expressed cardiac-specific proteins for alpha-actinin and troponin T. Among them, 1 X 10-7 mol/L icariin treatment resulted in a significantly advanced and increased mRNA level of alpha-cardiac myosin heavy chain (MHC) and myosin light chain 2v (MLC-2v) in EBs in the early cardiac developmental stage. Before shifting to the cardiomyocyte phenotype, icariin could evoke the accumulation of ES cells in G(0)/G(1) and accelerate apoptosis of the cell population (P < 0.05). Conclusion: Icariin facilitated the directional differentiation of ES cells into cardiomyocytes at a concentration of 1 X 10(-7) mol/L. The promoting effect of icariin on cardiac differentiation was related to increasing and accelerating gene expression of alpha-cardiac MHC and MLC-2v, as well as regulating the cell cycles and inducing apoptosis.