Resident c-kit(+) cells in the heart are not cardiac stem cells

Abstract

Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. Previously, cardiac c-kit(+) cells were reported to be CSCs with a potential to become myocardial, endothelial and smooth muscle cells in vitro and after cardiac injury. Here we provide further insights into the nature of cardiac c-kit(+) cells. By targeting the c-kit locus with multiple reporter genes in mice, we find that c-kit expression rarely co-localizes with the expression of the cardiac progenitor and myogenic marker Nkx2.5, or that of the myocardial marker, cardiac troponin T (cTnT). Instead, c-kit predominantly labels a cardiac endothelial cell population in developing and adult hearts. After acute cardiac injury, c-kit(+) cells retain their endothelial identity and do not become myogenic progenitors or cardiomyocytes. Thus, our work strongly suggests that c-kit(+) cells in the murine heart are endothelial cells and not CSCs.

Figure 1. Cardiac c-kit expression in c-kit^{H2B-tdTomato/+}mice.

(a) Diagram of the c-kit^{H2B-tdTomato/+}knock-in allele. (b–i) Sections of c-kit^{H2B-tdTomato/+}hearts at embryonic days (E) 8.5, 9.5, 12.5 and 14.5 (b–e) and at postnatal (P) days 1, 30, 60 and 120 (f–i). c2, e2, g2 and i2 are high-magnification images (without DAPI) of the areas outlined in c1, e1, g1 and i1, respectively. c-kit^H2B-tdTomato cells are denoted by arrows. LA, left atria; LV, left ventricle; OFT, outflow tract; RA, right atria; RV, right ventricle; VS, ventricular septum. n=3 for each stage. Scale bar, 100 μm.

Figure 2. Cardiac c-kit^H2B-tdTomato cells are PECAM⁺ endothelial cells.

(a,b) At E8.5 and E9.5, c-kit^H2B-tdTomato cells are endocardial (PECAM⁺). (c–f) c-kit^H2B-tdTomato cells express PECAM at E16.5 (c) and at P1–120 (d–f). Arrows indicate PECAM⁺ and tdTomato⁺ double-positive cells. Arrowheads indicate PECAM⁺ and tdTomato⁻ cells. (g,h) Cardiac smooth muscle cells (α-SMA⁺) are tdTomato⁻ at P120 (arrowheads). a2–h2 are high-magnification images of the areas outlined in a1–h1 (without DAPI), respectively. n=3 for each stage. Scale bar, 100 μm.

Figure 3. c-kit^nlacZ cells are of a Tie2 endothelial lineage.

(a) Diagram of the c-kit^{nlacZ-H2B-GFP/+}reporter allele (a1). The c-kit^H2B-GFP/+ allele is generated when the nlacZ cassette is removed by Cre excision (a2). (b–k) X-gal staining of c-kit^{nlacZ-H2B-GFP/+} and c-kit^{nlacZ-H2B-GFP/+};Tie2^Cre littermate hearts at E15.5 (b,c, sections) and at P1–90 (d–k). Arrows indicate comparable regions to X-gal⁺ or X-gal⁻ staining. Arrowheads indicate rare X-gal⁺ cells on c-kit^{nlacZ-H2B-GFP/+};Tie2^Cre hearts, suggesting that most c-kit⁺ cells lose the nlacZ gene because they are in the Tie2^Cre lineage. f2–k2 are high-magnification images of the areas outlined in f1–k1, respectively. n=3–5 for each stage. Scale bar, 400 μm (black) and 200 μm (white).

Figure 4. Active c-kit endothelial expression and myogenic potential assayed by transient induction of Cre activity in c-kit^MerCreMer/+ mice.

(a) Diagram of the c-kit^MerCreMer/+ allele. c-kit^MerCreMer/+ animals were crossed to the ROSA26R^tdTomato reporter line to obtain c-kit^MerCreMer/+;ROSA26R^tdTomato/+. (b–e) Cre activity was transiently induced in c-kit^MerCreMer/+;ROSA26R^tdTomato/+ animals at P30, P60 and P90 by tamoxifen injection on days 1–3. Hearts were harvested on days 4 and 14. Many tdTomato⁺ cells (arrows in b2, d2 and e2) were detected in hearts at P34 (b1), P64 (d1) and P104 (e1). These tdTomato⁺ cells were PECAM⁺ (c2, arrows, P30→34). b2, d2 and e2 are high-magnification florescent images of the areas outlined in b1, d1 and e1 (bright field), respectively. (f) Diagram of the cTnT^{nlacZ-H2B-GFP/+}allele and lineage tracing using c-kit^MerCreMer/+;cTnT^{nlacZ-H2B-GFP/+}mice. Cre activity was transiently induced by tamoxifen injection for 4 days on days 1, 2, 3 and 5 (days 1 and 2 for E11.5). Samples were collected on day 7 (day 3 for E11.5). (g) cTnT^H2B-GFP cells were detected at E13.5, P37, P67 and P97 (arrows), with the total number in the whole heart noted at the upper right corner. Scale bar, 1 mm (black) and 100 μm (white).

Figure 5. c-kit⁺ cells do not co-express Nkx2.5 in the injured region.

(a) Diagram of LAD ligation. (b) Masson trichrome staining shows the infarcted region of a c-kit^{H2B-tdTomato/+};Nkx2.5^H2B-GFP/+heart at 60 days post-surgery (dps). b1 and b2 are high-magnification images of the numbered outlined areas in b. (c–f) No c-kit^H2B-tdTomato/Nkx2.5^H2B-GFP double-positive cells were found in the infarcted regions at 3 (c), 21 (d), 30 (e) and 60 dps (f). c1/c2, d1/d2, e1/e2, and f1/f2 are high-magnification images of the numbered outlined areas in c, d, e and f, respectively. Scale bar, 500 μm (black) and 50 μm (white).

Figure 6. Cell type and lineage of c-kit⁺ cells in the injured heart.

(a) c-kit^H2B-GFP-positive cells were present in the infarcted region of Tie2^Cre;c-kit^{nlacZ-H2B-GFP/+} hearts at 30 dps. a2 is green channel of a1, and a3 is high-magnification image of the area outlined in a2. (b) Masson trichrome staining of cTnT^MerCreMer/+;c-kit^{nlacZ-H2B-GFP/+};ROSA26R^tdTomato/+ hearts at 60 dps shows the infarcted region. (c) Adjacent section of b. ROSA26R^tdTomato signal indicates myocardial cells after tamoxifen induction (c1). No c-kit^H2B-GFP cells were observed in the infarcted zone (arrows). c2 is green channel of c1. (d) Masson trichrome staining of c-kit^MerCreMer/+;cTnT^{nlacZ-H2B-GFP/+} hearts at 60 dps. (e) Adjacent section of d shows a few cTnT^H2B-GFP cells (<20) that were found in the infarcted zone (e1, arrowhead). cTnT^H2B-GFP cells were also present in a remote, uninjured region (e2, arrowhead). Scale bar, 100 μm.