Genetic Lineage Tracing of Sca-1+ Cells Reveals Endothelial but Not Myogenic Contribution to the Murine Heart

Abstract

Background: The adult mammalian heart displays a cardiomyocyte turnover rate of ≈1%/y throughout postnatal life and after injuries such as myocardial infarction (MI), but the question of which cell types drive this low level of new cardiomyocyte formation remains contentious. Cardiac-resident stem cells marked by stem cell antigen-1 (Sca-1, gene name Ly6a) have been proposed as an important source of cardiomyocyte renewal. However, the in vivo contribution of endogenous Sca-1⁺ cells to the heart at baseline or after MI has not been investigated.

Methods: Here we generated Ly6a gene-targeted mice containing either a constitutive or an inducible Cre recombinase to perform genetic lineage tracing of Sca-1⁺ cells in vivo.

Results: We observed that the contribution of endogenous Sca-1⁺ cells to the cardiomyocyte population in the heart was <0.005% throughout all of cardiac development, with aging, or after MI. In contrast, Sca-1⁺ cells abundantly contributed to the cardiac vasculature in mice during physiological growth and in the post-MI heart during cardiac remodeling. Specifically, Sca-1 lineage-traced endothelial cells expanded postnatally in the mouse heart after birth and into adulthood. Moreover, pulse labeling of Sca-1⁺ cells with an inducible Ly6a-MerCreMer allele also revealed a preferential expansion of Sca-1 lineage-traced endothelial cells after MI injury in the mouse.

Conclusions: Cardiac-resident Sca-1⁺ cells are not significant contributors to cardiomyocyte renewal in vivo. However, cardiac Sca-1⁺ cells represent a subset of vascular endothelial cells that expand postnatally with enhanced responsiveness to pathological stress in vivo.

Keywords: cell lineage; endothelial cells; myocardial infarction; regeneration; stem cells.

Figure 1:. Cardiac Sca-1⁺ Cells Contribute to the Vasculature Throughout Postnatal Growth.

A. Experimental scheme and timeline for genetic lineage tracing studies in this figure using constitutive Sca-1 (gene name Ly6a) Cre gene-targeted mice. B. Representative confocal micrographs of histological sections from hearts of Ly6a^+/Cre × R26-TdT mice at either postnatal day 1, 1.5 months of age, or 3 months of age, showing continual expansion of TdTomato⁺ cells (red) in the heart. Scale bars = 100 μm. C, D. Immunohistochemistry on cardiac histological sections was performed to detect CD31 (white) along with endogenous TdTomato fluorescence (red). DAPI (blue) was used to visualize nuclei. TdTomato⁺ endothelial cells were seen in small clusters at postnatal day 1 (C) and throughout the heart at 1.5 months of age (D). Scale bars = 10 μm. E, F. Representative flow cytometry plots (E) and quantitation (F) from dissociated Ly6a^+/Cre × R26-TdT mouse hearts at 3 months of age (n=3) analyzed using antibodies against Sca-1 and CD31. The first plot shows Sca-1 positivity by fluorochrome-conjugated antibody staining (Sca-1), versus CD31 positivity also by antibody (CD31). The second plot shows TdTomato⁺ cells (endogenous TdTomato fluorescence) versus side scatter within the Sca-1⁺ CD31⁻ gate (lower right quadrant) shown in the first plot as indicated. The final plot shows TdTomato⁺ cells (endogenous TdTomato fluorescence) versus side scatter within the Sca-1⁺ CD31⁺ gate (upper right quadrant) shown in the first plot as indicated.

Figure 2:. Cardiac Sca-1⁺ Cells Contribute Few Cardiomyocytes Throughout Postnatal Growth.

A. Experimental scheme and timeline for genetic lineage tracing studies in this figure using Ly6a-Cre gene-targeted mice. B. Immunohistochemistry was performed on cardiac histological sections from Ly6a^+/Cre × R26-TdT mice at 3 months of age using antibodies against sarcomeric α-actinin (white) and PCM1 (purple). DAPI (blue) was used to visualize nuclei. Representative confocal micrograph shows a rare TdTomato⁺ cardiomyocyte (yellow box). Scale bar: 100 μm. C. High-magnification confocal micrographs of the yellow boxed area denoted in B. Yellow arrowhead indicates a TdTomato⁺ cardiomyocyte (α-actinin⁺ PCM1⁺). Scale bars = 10 μm. D. Quantitation of TdTomato⁺ cardiomyocytes from histological sections of hearts from Ly6a^+/Cre × R26-TdT mice at 3 months of age. Quantitation is shown from hearts of n=5 mice over 144 histological sections and 303260 total cardiomyocytes counted. E, F. Representative flow cytometry plots (E) and quantitation (F) from the bone marrow of Ly6a^+/Cre × R26-TdT mice at 3 months of age (n=3). The first plot shows forward (FSC-A) versus side (SSC-A) scatter to determine size distribution of the bone marrow. The second plot shows TdTomato⁺ cells (endogenous TdTomato fluorescence) versus side scatter from the gate shown in the first plot as indicated (E). Cells within this second gate were stained with antibodies against Sca-1 and CD45, which showed primarily mature CD45⁺ leukocytes that were TdTomato⁺ (F).

Figure 3:. Inducible Genetic Lineage Tracing in the Adult Mouse Tracks Sca-1⁺ Cells During Aging.

A. Experimental scheme for genetic lineage tracing studies in this figure using a tamoxifen-inducible Ly6a-MerCreMer gene-targeted mouse line and the R26-eGFP reporter line. B. eGFP reporter expression is dependent on tamoxifen induction, as untreated Ly6a^+/MerCreMer × R26-eGFP mice aged for one year do not show eGFP⁺ cells across multiple tissues surveyed. C. Ly6a^+/MerCreMer × R26-eGFP mice treated with tamoxifen starting at adulthood (3 mos) out to 1 yr of age showed endothelial cell labeling (CD31, red) of eGFP⁺ cells (green) throughout the heart, lung, liver, intestine, and kidney. Yellow boxes indicate areas shown to the right of each image in an enlarged view. Other cell types previously reported to express Sca-1 are also labeled with eGFP. DAPI (blue) was used to visualize nuclei. Scale bars = 100 μm.

Figure 4:. Sca-1⁺ Cells Contribute to the Vasculature in the Murine Heart During Adulthood and After Injury.

A. Experimental scheme for genetic lineage tracing studies in this figure using Ly6a-MerCreMer mice along with the R26-eGFP reporter line during aging (Timeline #1) or post-MI (Timeline #2). B, C. Representative confocal micrographs (B) showing co-localization of CD31 (red) and eGFP (green) in cardiac histological sections in Ly6a^+/MerCreMer × R26-eGFP mice continually treated with tamoxifen from 3 months to 1 year of age (9 m of labeling). Scale bars = 100 μm. Yellow box denotes area shown in enlarged view in (C), demonstrating eGFP⁺ capillaries and small vessels adjacent to eGFP⁻ large vessels. Scale bar = 100 μm. D. Quantitation of eGFP⁺ cells from dissociated Ly6a^+/MerCreMer × R26-eGFP hearts (n=4) by flow cytometry using antibodies against Sca-1 and CD31. E. Quantitation of eGFP⁺ cardiomyocytes from hearts of Ly6a^+/MerCreMer x R26-eGFP mice over 9 m of tamoxifen labeling in adulthood. Quantitation was from n=4 mice and 374832 total cardiomyocytes counted from histological sections. F-I. Ly6a^+/MerCreMer × R26-eGFP mice were given tamoxifen for 4 weeks followed by MI injury. Representative high-magnification confocal micrographs of heart histological sections labeled with CD31 (red) and DAPI (blue) are shown prior to MI (F) or at 2 wks post-MI (G) and at 8 wks post-MI (H). Yellow arrowhead in H indicates a vascular branching event by previously pulse-labeled eGFP⁺ endothelial cells. Scale bars = 10 μm. I. Quantitation of overall cardiac capillary density and the percentage of CD31⁺ endothelial cells labeled with eGFP in Ly6a^+/MerCreMer x R26-eGFP mice post-MI. Green bars represent the fraction of CD31⁺ endothelial cells that were eGFP⁺, relative to total endothelial cells (green plus blue bars). n=4–5 mice per time point.