Fate Mapping of Sca1 + Cardiac Progenitor Cells in the Adult Mouse Heart

Sca1 + cardiac progenitor cells have been reported as resident cardiac stem cells that can differentiate into cardiomyocytes.1,2 Whether endogenous Sca1+ cells contribute to cardiomyocytes in vivo remains unclear. To trace Sca1+ cardiac progenitor cells, we generated a Sca1-2A-CreER knockin mouse line that harbors CreER inframe with Sca1 coding region by a 2A self-cleaving peptide sequence (Figure, A). All mice protocols were approved by the Institutional Animal Care and Use Committee at the Institute for Nutritional Sciences and the Institute of Biochemistry and Cell Biology. To characterize whether Sca1-2A-CreER targets endogenous Sca1+ cells, we performed pulse tamoxifen induction and collected heart samples within 24 to 48 hours after tamoxifen induction. Flow cytometric analysis and immunostaining data showed that Sca1-2A-CreER specifically labels endogenous Sca1+ cells in the mouse heart (Figure, B through D). Z-stack confocal images showed that Sca1+ cells were VE-cad+ cells (Figure, E), and quantitatively the majority of VE-cad+ endothelial cells (67.48±2.18%) were tdTomato+, whereas almost all tdTomato+ cells (95.14±0.71%) were VE-cad+ endothelial cells. Immunostaining for tdTomato and TNNI3 showed no labeling of cardiomyocytes by Sca1-2A-CreER (Figure, F). We also did not find any tdTomato+ cells in the dissociated cardiomyocytes (Figure, G). We next used Sca1-2A-CreER to perform fate-mapping analysis of Sca1+ cells during cardiac homeostasis. Sca1-2A-CreER;R26-tdTomato hearts were collected at 12 weeks after tamoxifen induction. Z-stack confocal images of heart sections stained with TNNI3 and tdTomato showed no tdTomato+ cardiomyocytes (Figure, H). Flow cytometric analysis showed no tdTomato+ cardiomyocytes (Figure, I). Dissociated cardiomyocytes also exhibited no tdTomato+ signal (Figure, J). By contrast, flow cytometric analysis showed that 60.88±2.38% endothelial cells expressed tdTomato (Figure, K). It is notable that 94.25±0.54% tdTomato+ cells were CD31+ endothelial cells (Figure, K), indicating that most Sca1+ cells adopted an endothelial cell fate during cardiac homeostasis. Immunostaining for tdTomato and CD31 on heart sections showed that Sca1+ cells contributed to a substantial number of endothelial cells (Figure, L). Sca1+ cells also contribute to cardiac fibroblasts. Flow cytometric analysis showed that 1.79±0.14% PDGFRa+ cells were labeled by tdTomato, whereas 0.52±0.072% tdTomato+ cells expressed PDGFRa (Figure, M). Z-stack confocal images of heart sections stained with tdTomato and PDGFRa showed that 1.82±0.11% of PDGFRa+ cells were labeled by tdTomato, whereas 0.51±0.025% of tdTomato+ cells were PDGFRa+ in the normal heart during homeostasis (Figure, N). To test whether Sca1+ cells generated new cardiomyocytes after cardiac injury, we performed myocardial infarction (MI) model at 2 weeks after tamoxJuan Tang, PhD Yan Li, PhD Xiuzhen Huang, BA Lingjuan He, PhD Libo Zhang, BS Haixiao Wang, BS Wei Yu, PhD Wenjuan Pu, PhD Xueying Tian, PhD Yu Nie, PhD Shengshou Hu, MD, PhD Qing-Dong Wang, PhD Kathy O. Lui, PhD Bin Zhou, MD, PhD