title: The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells.
abstract: BACKGROUND
  Recent studies have reported that microRNA-145 (miR-145) is a critical mediator in the regulation of proliferation, differentiation, and phenotype expression of smooth muscle cells (SMCs). Previously, we established a system for differentiating human ESCs into vascular cells including endothelial cells (ECs) and vascular smooth muscle cells (SMCs). In the present study, we investigated the role of miR-145 in the differentiation process from human ESCs into ECs and SMCs.
  
  
  RESULTS
  Undifferentiated human ESCs were induced to differentiate into vascular lineage according to our established method. Quantitative RT-PCR analysis revealed that human ESC-derived precursor of SMCs (ES-pre-SMCs), similar to human aortic SMCs, expressed a significant amount of miR-145 as well as smooth muscle-specific proteins, compared to undifferentiated human ESCs, adult ECs, or ESC-derived ECs (ES-ECs). However, morphological analysis revealed that human ES-pre-SMCs appeared round and flattened in shape, though human aortic SMCs exhibited the typical spindle-like morphology of SMCs. In addition, Krüppel-like factor 4 and 5 (KLF4 and 5), direct targets of miR-145 and suppressors of smooth muscle differentiation, were upregulated in ES-pre-SMCs compared to aortic SMCs, indicating ES-pre-SMCs were not fully differentiated SMCs. Overexpression of miR-145 in ES-pre-SMCs upregulated the expression of smooth muscle markers, repressed KLF4 and 5 expressions, and changed their morphology into a differentiated spindle-like shape. Furthermore, by introduction of miR-145, ES-pre-SMC proliferation was significantly inhibited and carbachol-stimulated contraction of ES-pre-SMCs was significantly increased. In contrast, downregulation of miR-145 in ES-pre-SMCs upregulated KLF4 and 5 expressions, suppressed the expression of smooth muscle markers, and left unchanged their proliferation and contractility. In ES-ECs, miR-145 overexpression did not induce the synthesis of smooth muscle-related proteins nor suppress the expression of endothelial nitric oxide synthase.
  
  
  CONCLUSIONS
  We showed that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs as they become fully differentiated SMCs. Overexpression of miR-145 on human ES-pre-SMCs is a promising method to obtain functional mature SMCs from human ESCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases.
authors.affiliation: Division of Endocrinology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: Kenichi
authors.initials: K
authors.lastname: Yamahara
authors.forename: Koichiro
authors.initials: K
authors.lastname: Homma
authors.forename: Sayuri
authors.initials: S
authors.lastname: Suzuki
authors.forename: Shizuka
authors.initials: S
authors.lastname: Fujii
authors.forename: Ryuji
authors.initials: R
authors.lastname: Morizane
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Monkawa
authors.forename: Yumi
authors.initials: Y
authors.lastname: Matsuzaki
authors.forename: Kenji
authors.initials: K
authors.lastname: Kangawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Itoh
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation; D048430:Cell Shape; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D005838:Genotype; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D010641:Phenotype; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D015415:Biomarkers; C000715467:KLF4 protein, human; C492214:KLF5 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C541010:MIRN145 microRNA, human; D035683:MicroRNAs; D002217:Carbachol