LAUSR

De novo cardiomyogenesis is limited to ≈1% per year in the adult mammalian heart.1 Whether newly formed cardiomyocytes are derived from division of pre-existing myocytes or from differentiation of resident cardiac progenitor cells is a topic of debate. Cardiac progenitor cells have been posited as a source of endogenous cardiomyocyte renewal and as cells that can be harvested, expanded in vitro, and delivered therapeutically after infarction. Stem cell antigen 1 (Sca-1), which was initially described as a surface marker of murine hematopoietic stem cells, has been reported to mark resident cardiac progenitor cells,2 and a previous study reported frequent contribution of Sca-1–expressing cells to cardiomyocytes.3 However, the transgenic approach used in this study resulted in more widespread expression than just Sca-1–expressing cells, complicating interpretation of the results.4 Because more recent fate mapping studies questioned the existence of resident cardiac progenitor cells, we aimed to test the hypothesis that endogenous Sca-1–expressing cells are progenitors for cardiomyocytes in vivo under physiological and pathophysiological conditions. We generated a tamoxifen-inducible genetic lineage-tracing Sca-1 mER-CremER knock-in mouse model (Sca-1mCm/+)5 and cross-bred to a Rosa26 tdTomato reporter line (Sca-1mCmR26tdTomato) to assess the fate of cellular descendants of Sca1–expressing cells in vivo. Adult Sca-1mCmR26tdTomato animals were treated with tamoxifen daily for 7 days, and hearts were harvested 7 days after the last tamoxifen injection to assess recombination. Recombination in the heart averaged 33% in Sca-1+/CD31+ populations and 13% in the Sca-1+/CD31– population (Figure [A] and [B]). No tdTomato+ cells were identified by flow cytometry in Sca-1mCmR26dTomato mice that did not receive tamoxifen. Histology (Figure [C]) demonstrated a mostly endothelial expression pattern in Sca-1mCmR26dTomato hearts that colocalized with CD31 expression. Additionally, there was overlap between Sca-1 and tdTomato staining as expected, as well as some NG2-expressing perivascular cells that overlapped with tdTomato. In tissue sections, TdTomato was not identified in cardiomyocytes. Additionally, tamoxifen-dependent tdTomato expression in tissues known to express Sca-1 (kidney, lung, liver, ileum) confirmed the presence of tdTomato+ cells, predominately in an endothelial pattern; this was absent in tissue from animals that received no tamoxifen (data not shown). Next, we set out to test whether new cardiomyocytes were derived from descendants of Sca-1–expressing cells after myocardial infarction (MI). Adult Sca-1mCmR26dTomato mice were treated with tamoxifen daily for 7 days, followed by a 7-day chase period. The mice then underwent MI or were allowed to age normally. Mice were euthanized 6 months later for examination of the heart. We enzymatically dissociated cardiomyocytes and quantified either fraction of tdTomato+ cardiomyocytes from Lauren E. Neidig, DVM* Florian Weinberger, MD* Nathan J. Palpant, PhD John Mignone, MD, PhD Amy M. Martinson, BS Daniel W. Sorensen, BS Ingrid Bender, BS Natsumi Nemoto, BS Hans Reinecke, PhD Lil Pabon, PhD Jeffery D. Molkentin, PhD Charles E. Murry, MD, PhD† Jop H. van Berlo, MD, PhD†