Abstract 10657: Microrna-200c and -150 Play a Role in Human Embryonic Stem Cell Differentiation Into Endothelial Cells and Blood Vessel Formation by Targeting Transcription Repressor Zeb1
Background: There is evidence suggesting the regulation of microRNAs (miRNAs) in a myriad of vascular biology events such as cardiogenesis, angiogenesis or arteriogenesis.
Objective: We investigate the role of miRNA in controlling human embryonic stem cell (hESC) fate in differentiation towards the endothelial lineage and chick embryonic blood vessel formation.
Methods and Results: Clumps of undifferentiated hESCs were firstly cultured on matrigel coated flasks and in endothelial differentiation medium to initial endothelial cell (EC) differentiation. At day 12 of differentiation, flow cytometry analysis showed that ∼34% of the heterogeneous differentiated hESCs were CD146 positive. This population of cells was further cell-sorted for CD146 and expanded in vitro. The in-vitro expanded CD146+ cells were positive for EC markers, capable of ac-LDL uptake, binding to Lectin and formation of vascular structures in vitro and in vivo, suggesting that these expanded CD146+ cells are functional ECs. Determination of the potential role of miRNA involved in early endothelial development was next carried out using miRNA array expression profiling in the differentiating hESCs. Potential up-regulated miRNA were selected from the miRNA array analysis and further analysis found that miR-150 and miR-200c were crucial in vascular endothelial lineage differentiation. Transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) was identified as the common target gene of miRNA-200C and -150 by using wild type and miRNA binding sites mutated luciferase reporters and luciferase activity analyses. It was found that inhibition of ZEB1 was required for miRNA-200C mediated EC gene expression. Finally, we also demonstrated for the first time that miRNA-200c and -150 play an important role in chick embryonic blood vessel formation by in vivo inhibition of miRNA-200C or -150 in the developing chick embryos.
Conclusions: In conclusion, we have established an effective model to derive ECs in vitro and demonstrated the involvement of miR-150 and miR-200c in hESCs differentiation towards the EC lineage and chick embryonic arteriogenesis. This study implicates a potential benefit for stem cell therapy in future.
- © 2012 by American Heart Association, Inc.