Regulated Expression and Role of c-Myb in the Cardiovascular-Directed Differentiation of Mouse Embryonic Stem Cells

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)alpha. By day 3.75, hematopoesis-capable VEGFR2+ cells were fewer, whereas cardiomyocyte-directed VEGFR2+/PDGFR alpha+ 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+/PDGFR alpha+ 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 PDGFR alpha, 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 (RA) as compared with VEGFR2+/PDGFR alpha+ cells. By contrast, VEGFR2+/PDGFR alpha+ cells cultured without RA beat spontaneously, like cardiomyocytes between days 7 and 14, and expressed cardiac troponin. Notably, RA was required to more fully differentiate SMC from VEGFR2+ cells and completely blocked differentiation of cardiomyocytes from VEGFR2+/PDGFR alpha+ cells. Conclusions: c-Myb is tightly regulated by proteosomal degradation during cardiovascular-directed differentiation of ESC, expanding early-stage VEGFR2+ progenitors capable of RA-responsive SMC formation. (Circ Res. 2012;110:253-264.)