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Regulated Expression and Role of c-Myb in the Cardiovascular-Directed Differentiation of Mouse Embryonic Stem Cells.
MedLine Citation:
PMID:  22116818     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Rationale:c-myb null (knockout) embryonic stem cells (ESC) can differentiate into cardiomyocytes but not contractile smooth muscle cells (SMC) in embryoid bodies (EB).Objective:To define the role of c-Myb in SMC differentiation from ESC.Methods and Results:In wild-type (WT) EB, high c-Myb levels on days 0-2 of differentiation undergo ubiquitin-mediated proteosomal degradation on days 2.5-3, resurging on days 4-6, without changing c-myb mRNA levels. Activin-A and bone morphogenetic protein 4-induced cardiovascular progenitors were isolated by FACS for expression of vascular endothelial growth factor receptor (VEGFR)2 and platelet-derived growth factor receptor (PDGFR)α. By day 3.75, hematopoesis-capable VEGFR2+ cells were fewer, whereas cardiomyocyte-directed VEGFR2+/PDGFRα+ cells did not differ in abundance in knockout versus WT EB. Importantly, highest and lowest levels of c-Myb were observed in VEGFR2+ and VEGFR2+/PDGFRα+ cells, respectively. Proteosome inhibitor MG132 and lentiviruses enabling inducible expression or knockdown of c-myb were used to regulate c-Myb in WT and knockout EB. These experiments showed that c-Myb promotes expression of VEGFR2 over PDGFRα, with chromatin immunopreciptation and promoter-reporter assays defining specific c-Myb-responsive binding sites in the VEGFR2 promoter. Next, FACS-sorted VEGFR2+ cells expressed highest and lowest levels of SMC- and fibroblast-specific markers, respectively, at days 7-14 after retinoic acid as compared with VEGFR2+/PDGFRα+ cells. By contrast, VEGFR2+/PDGFRα+ cells cultured without RA beat spontaneously, like cardiomyocytes between days 7 and 14, and expressed cardiac troponin. Notably, retinoic acid was required to more fully differentiate SMC from VEGFR2+ cells and completely blocked differentiation of cardiomyocytes from VEGFR2+/PDGFRα+ cells.Conclusions:c-Myb is tightly regulated by proteosomal degradation during cardiovascular-directed differentiation of ESC, expanding early-stage VEGFR2+ progenitors capable of retinoic acid-responsive SMC formation.
Authors:
Masayoshi Ishida; Omar El-Mounayri; Steven Kattman; Peter Zandstra; Hiroshi Sakamoto; Minetaro Ogawa; Gordon Keller; Mansoor Husain
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-11-23
Journal Detail:
Title:  Circulation research     Volume:  -     ISSN:  1524-4571     ISO Abbreviation:  -     Publication Date:  2011 Nov 
Date Detail:
Created Date:  2011-11-25     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0047103     Medline TA:  Circ Res     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, and the McEwen Centre for Regenerative Medicine, Toronto General Hospital Research Institute, Toronto, Canada; and the Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan.
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