Abstract 20873: A Multipotent Cardiovascular Progenitor Population Identified by the SRF Cardiac-Specific Enhancer is Detected Prior to Mesoderm Formation
Cardiac progenitors hold appeal for repair of diseased hearts because they are highly proliferative and can potentially generate all major cell types in the adult heart. Serum response factor (SRF) is a transcription factor expressed prior to appearance of mesoderm and is necessary for gastrulation. Although SRF is expressed in multiple tissues, we identified an enhancer sequence that confines expression to the cardiac lineage. To test the hypothesis that this SRF cardiac enhancer can be used to isolate multi-potent cardiac progenitors (SRFGFP cells), we generated SRFGFP cells using a construct consisting of the SRF promoter and cardiac enhancer flanking eGFP. This was targeted to the HRPT locus in mouse ES cells using an inducible cassette exchange strategy with Cre recombinase. Positive SRFGFP clones, confirmed by RT-PCR, were differentiated in embryoid bodies (EBs) to monitor GFP expression. GFP expression was first observed at 8 hours. RT-qPCR confirmed that GFP+ cells from day 1 EBs express SRF but not early mesoderm markers brachyury (T) or Mesp1, indicating that these cells are detectable prior to mesoderm formation. Importantly, SRFGFP cells do not express pluripotency markers Oct4 or Nanog, thus are not residual ES cells. Multiphoton microscopy demonstrated that SRFGFP cells proliferate in permissive areas of live EBs, forming localized clusters by day 4 and ultimately GFP+ contracting regions. Flow cytometry analysis of day 2–5 EBs revealed that 3–6% of cells express GFP. Isolated day 2 SRFGFP cells have the capacity to differentiate in vitro into cell types found within the adult heart, including cardiac myocytes (anti-cTnT), endothelial cells (anti-CD31), fibroblasts (anti-fibroblast) and smooth muscle cells (anti-sm-MHC). SRFGFP cells isolated from day 4 EBs express markers of cardiac precursors, including Nkx2.5, Flk1, Isl1, T and Tbx18. SRFGFP cells, when serially passaged, maintain expression of cardiac progenitor proteins and differentiation capacity, introducing the possibility of expansion pre-transplantation. These results demonstrate a reporter system to identify and isolate cardiac progenitors specified prior to mesoderm formation, and this early population of progenitors holds unique promise for cardiac repair.
- © 2010 by American Heart Association, Inc.