Abstract 12581: Epigenetic Activation of Vascular Genes Enhances Endothelial Cell Differentiation: The Role of MicroRNA-495 Inhibition in hiPSCs
Background and Objective: miR-495 inhibition is known to enhance mesoderm differentiation, but its role in endothelial cell (EC) differentiation is not clear. The present study seeks to understand if miR-495 inhibition promotes EC differentiation of human induced pluripotent stem cells (hiPSCs) and investigates mechanisms underlying its potential to activate vascular genes.
Methods: The knock down of miR-494 in hiPSCs via lentivirus (hiPSCanti-495, hiPSCScr as control) was confirmed by qPCR. The expression of pluripotent markers and the mesoderm marker (Brachyury) in hiPSCs was also analyzed by qPCR. EC differentiation of hiPSCs was induced via embryoid body (EB) formation. Immunostaining was used to determine the expression of vascular genes (CD31 and CDH5). The methylation status of CD31 and CDH5 promoters was analyzed by sodium bisulfite sequencing. Finally, CD31 and CDH5 promoters localized with histone and posttranslational modifications (H3K4me3, H3k27me3, and Acetyl-H3K9) were analyzed by ChIP-qPCR.
Results: The expression of pluripotent markers was not influenced by miR-495 knockdown in undifferentiated hiPSC, while Brachyury was increased in EBs derived from hiPSCanti-495 (Fig. 1). CD31 and CDH5 were expressed in hiPSCanti-495 which showed typical endothelial morphology. MiR-495 inhibition induced demethylation of some CG base sites in the CD31 and CDH5 promoters, but not significantly as compared to hiPSCScr. The relative levels of H3K4me3 and acetyl-H3K9 (relative to transcriptional activation) of CD31 and CDH5 promoters were significantly increased in hiPSCanti-495.
Conclusion: miR-495 represents a promising potential mediator for promoting EC differentiation of hiPSC by epigenetically regulating vascular genes.
Author Disclosures: J. Liang: None. W. Huang: None. W. Cai: None. M. Xu: None. R.W. Millard: None. Y. Wang: None.
- © 2014 by American Heart Association, Inc.