To investigate the role of miRNA in controlling human embryonic stem (hES) cell differentiation toward the endothelial lineage and chick embryonic blood vessel formation, undifferentiated hES cells were first cultured on Matrigel-coated flasks and in endothelial cell growth medium-2 (EGM-2) to initiate endothelial cell (EC) differentiation. CD146(+) cells were isolated from differentiating hES cells and expanded in vitro. The in vitro expanded CD146(+) cells were positive for EC markers, capable of Ac-LDL uptake, lectin binding, and the formation of vascular structures in vitro and in vivo. miRNA gain/loss-of-function analyses revealed that miR-150 and miR-200c were crucial in EC differentiation. Transcriptional repressor zinc finger E-box-binding homeobox 1 (ZEB1) was identified as the communal target gene of miRNA-200C and -150, and inhibition of ZEB1 was required for miRNA-200C or -150 mediated EC gene expressions. Moreover, we demonstrated that ZEB1 could transcriptionally repress EC gene expression through direct binding to promoters of EC genes. Finally, we also demonstrated that miRNA-200c and -150 played an important role in chick embryonic blood vessel formation by in vivo inhibition of miRNA-200C or -150 in developing chick embryos, and blocking ZEB1 signaling in CD146-positive cells could rescue the inhibitory effects of miR-200c inhibiton in in vivo vasculogenesis. Our findings revealed that miR-150 and miR-200c play an important role in human endothelial lineage specification and chick embryonic vasculogenesis by targeting ZEB1.
Keywords: Cell differentiation; Endothelial cells; MicroRNAs; Stem cells; Vasculogenesis; Zinc finger E-box-binding homeobox 1; miR-150; miR-200C.
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