Abstract 14376: The Homeobox Transcription Factor, Irx4, Enables Identification and Purification of a Multipotent, Ventricular Myocardium-specific Cardiovascular Progenitor
Cardiac progenitors have been presented as potential cell therapeutics, due to their cardiogenic potency. Iroquois homeobox protein 4 (Irx4) is the earliest known marker of ventricular myocardium differentiation, and the transcription factor is restricted to the ventricles throughout embryogenesis and into adulthood. Our goal is to identify a multipotent, ventricular-specific cardiac progenitor, and to do so we have selected Irx4 as a genetic marker.
We have targeted the Irx4 locus in R1 mESCs using a recombineering approach to insert fluorescence (tdTomato), bioluminescence (luciferase), and antibiotic resistance (hph) cassettes at the 3’ end of the gene. Six mESC clones were properly targeted Irx4luc-tdTom-hph/wt ES cells. RT-PCR, western blot analysis, and immunofluorescence assays demonstrated the functional integration of the reporters during embryoid body (EB) differentiation. Following 4 days of differentiation of the Irx4luc-tdTom-hph/wt cells in EBs, selection with hygromycin was carried out for 2 days, and day 6 cells were plated onto STO cell feeder layers for expansion. Selected Irx4+ cells are proliferative, expressing the cell cycle antigen, Ki67, and could be passaged more than 12 times. The Irx4+ cells express cKit, Flk1, and CXCR4 on the cell surface. Selected Irx4+ cells demonstrate cardiovascular potency when re-aggregated and differentiated in hanging drops resulting in ventricular myocyte-enriched cell preparations (65±3.7% cTnT+; 67±2.6% Myl2+, 22±3.1% SmMHC+, and 6±2% CD31+ cells). Myl2 and SmMHC have been established as reliable markers of ventricular myocytes, and smooth muscle cells, respectively. Also, CD31 is specific for immature endothelial cells, making this an ideal marker to identify endothelial progeny. Given the spatial expression of Irx4 during cardiogenesis, and the ventricular phenotype of the myocyte progeny, it is reasonable to suggest that Irx4+ progenitors contribute significantly to ventricular myocardium differentiation. The selected Irx4+ population represents the first ventricular-specific multipotent progenitor population identified and holds promise for generating the cardiovascular lineages necessary to reconstitute infarcted myocardium.
- © 2013 by American Heart Association, Inc.