C-Myb–Dependent Smooth Muscle Cell Differentiation

Abstract

Both in vitro and in vivo studies have implicated the c-Myb transcription factor in vascular smooth muscle cell (SMC) proliferation and hematopoiesis. However, its role in differentiation and maturation of contractile, as opposed to proliferating, SMCs has not been investigated. Here we demonstrate that c-myb(-/-) embryonic stem cells (ESCs) are incapable of producing embryoid bodies (EBs) with spontaneously contracting SMCs but can differentiate into contracting cardiomyocytes unimpaired. Quantitative real-time RT-PCR revealed that whereas mesodermal differentiation was unaffected, myocardin, a critical determinant of SMC differentiation, became upregulated at day 7 in wild-type, but not in c-myb(-/-) EBs. SMC-specific genes, smooth muscle alpha-actin, SM22alpha and smooth muscle myosin heavy chain reached peak expression levels by day 15 of differentiation and were 2- to 3-fold higher in wild-type as compared with c-myb(-/-) derived EBs. Similarly, fluorescence-activated cell-sorting analysis confirmed significantly different proportions of smooth muscle alpha-actin-positive cells in wild-type (26.8+/-0.7%) versus c-myb(-/-) (12.3+/-0.4%) EBs. Temporal induction of these SMC-specific markers preceded and paralleled contractile SMC appearance and predicted the relative (in)ability of c-myb(-/-) and wild-type ESC lines to generate EBs with contracting SMCs. Importantly, data from EBs faithfully predicted a significant reduction in c-myb(-/-) cell contribution to SMC lineage in vivo, in chimeric E11.5 embryo and adult aortas relative to brain and skin chimerism, respectively. Moreover, the visceral SMC population in chimeric embryos was nearly devoid of c-myb(-/-) cells. Our data are the first to implicate c-Myb in SMC differentiation from precursor stem cell-derived populations, reinforcing its potential role in phenotypic modulation of SMCs and vascular disease.