Endothelial Lineage Differentiation from Induced Pluripotent Stem Cells Is Regulated by MicroRNA-21 and Transforming Growth Factor β2 (TGF-β2) Pathways

Background: Induced pluripotent stem cells (iPSCs) constitute an attractive source of cells for regenerative medicine. Results: MicroRNA-21 mediates endothelial differentiation derived from iPSCs in presence of VEGF. Conclusion: MicroRNA-21 and TGF-2 signaling pathways regulate iPSC differentiation to endothelial lineage. Significance: Elucidation of the molecular mechanisms underlying microRNA-21-regulated iPSC differentiation might provide the basic information for stem cell therapy of vascular diseases. Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-2 during iPSC differentiation. Indeed, treatment of iPSCs with TGF-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGF-2, strongly decreased VE-cadherin expression. Furthermore, TGF-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.