Abstract 16067: c-Myb Levels Regulate Flk1+ Progenitors and Subsequent Smooth Muscle Differentiation From Mouse Embryonic Stem Cells.
Background: c-myb encodes the transcription factor c-Myb, which is involved in hematopoiesis (HP) and vascular smooth muscle cell (SMC) proliferation. However, a mechanism for why c-myb knockout (KO) mouse embryonic stem cells (ESC) cannot form contractile SMC in embryoid bodies (EB), but form contracting cardiomyocytes (CM) with greater efficiency than wild-type (WT) ESC, was not known. Flk1 and PDGFRα are surface markers used to identify early progenitors. Here we report on the regulated expression and role of c-Myb during SMC differentiation from specific progenitors.
Methods & Results: Western blot revealed high levels of c-Myb on d0-2 of differentiation in WT EB, with rapid and complete loss of c-Myb on d2.5-3, and re-expression on d4−6. Loss of c-Myb on d2.5-3 was not associated with changes in c-myb mRNA, but was completely blocked by the proteosome inhibitor MG132. ESC-derived cardiovascular progenitors induced by Activin-A and BMP4 underwent flow cytometry for Flk1 and PDGFRα. By d3.75, the appearance of HP-capable Flk1+/PDGFRα- (FSP), and to a lesser extent CM-directed Flk1+/PDGFRα+ (DP), progenitors was significantly impaired in KO vs. WT EB despite varying combinations of Activin-A and BMP4. Indeed, KO EB could not meaningfully generate FSP. In WT cells, Western blot revealed highest vs. lowest c-Myb levels in FSP vs. DP and Flk1-/PDGFRα+ (PSP) progenitors. MG132 and lentivirus enabling inducible over-expression or knockdown (shRNA) of c-myb were used to regulate c-Myb levels in WT and KO EB. Together, these experiments confirmed that higher levels of c-Myb promote Flk1+ over PDGFRα+. Next, flow-sorted cells were cultured in bFGF and VEGF (5ng/ml each) with and without retinoic acid (RA, 10nM). RA for 7-14d induced significantly greater numbers and levels of SMC-specific markers in FSP than in DP or PSP progenitors, in which levels of SMC-markers were low and comparable to the fibroblast marker DDR2.
Conclusions: c-Myb is tightly regulated by proteosomal degradation in differentiating mouse ESC prior to emergence of Flk1 and PDGFRα. c-Myb is necessary for the differentiation of FSP (typically HP) progenitors, which manifest high potential for SMC differentiation. Loss of FSP cells appears to be the basis for the inability of c-myb KO EB to form SMC.
- Stem/progenitor cells
- Cardiovascular development
- Smooth muscle
- Cardiac development
- Stem cell and atherosclerosis
- © 2010 by American Heart Association, Inc.