title: Endothelial lineage differentiation from induced pluripotent stem cells is regulated by microRNA-21 and transforming growth factor β2 (TGF-β2) pathways.
abstract: 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-β2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-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.
authors.affiliation: From the Cardiovascular Division, King's College London, British Heart Foundation Centre, London SE5 9NU, United Kingdom.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Di Bernardini
authors.forename: Paola
authors.initials: P
authors.lastname: Campagnolo
authors.forename: Andriana
authors.initials: A
authors.lastname: Margariti
authors.forename: Anna
authors.initials: A
authors.lastname: Zampetaki
authors.forename: Eirini
authors.initials: E
authors.lastname: Karamariti
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Hu
authors.forename: Qingbo
authors.initials: Q
authors.lastname: Xu
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation / Q000502:physiology; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D035683:MicroRNAs; D051059:PTEN Phosphohydrolase; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D051900:Smad3 Protein; D053781:Transforming Growth Factor beta2; D000099152:Cadherin 5; C531950:MIRN21 microRNA, mouse; C497791:Smad3 protein, mouse; C509042:Tgfb2 protein, mouse; C487757:Pten protein, mouse
keywords: Endothelial Cell; Induced Pluripotent Stem Cells; MicroRNA; Transforming Growth Factor Beta (TGFbeta); Vascular Endothelial Growth Factor (VEGF)