Cardiomyogenic potential of C-kit(+)-expressing cells derived from neonatal and adult mouse hearts

Abstract

Background: C-kit is a receptor tyrosine kinase family member expressed in hematopoietic stem cells. C-kit is also transiently expressed in cardiomyocyte precursors during development and in a rare cell population in the normal adult heart. In the present study, the cardiomyogenic potential of c-kit(+) cells isolated from normal neonatal, normal adult, and infarcted adult mouse hearts was evaluated.

Methods and results: Magnetic activated cell sorting was used to prepare c-kit(+) cells from the hearts of ACT-EGFP/MHC-nLAC double transgenic mice. These animals exhibit widespread enhanced green fluorescent protein (EGFP) expression and cardiomyocyte-restricted nuclear beta-galactosidase activity, thus permitting simultaneous tracking of cell survival and differentiation. A subset of the c-kit(+) cells from double transgenic neonatal hearts acquired a cardiomyogenic phenotype when cocultured with fetal cardiomyocytes (2.4% of all EGFP(+) cells screened) but rarely when cultured alone or when cocultured with mouse fibroblasts (0.03% and 0.05% of the EGFP(+) cells screened, respectively). In contrast, c-kit(+) cells from normal adult double transgenic hearts failed to undergo cardiomyogenic differentiation when cocultured with nontransgenic fetal cardiomyocytes (>18 000 EGFP(+) cells screened) or when transplanted into normal or infarcted adult mouse hearts (14 EGFP(+) grafts examined). A single c-kit(+) cell from an infarcted double transgenic adult heart was observed to acquire a cardiomyogenic phenotype in coculture (>37 000 EGFP(+) cells screened).

Conclusions: These data suggest that the ability of cardiac-resident c-kit(+) cells to acquire a cardiomyogenic phenotype is subject to temporal limitations or, alternatively, that the cardiomyogenic population is lost. Elucidation of the underlying molecular basis may permit robust cardiomyogenic induction in adult-derived cardiac c-kit(+) cells.

Figure 1. Characterization c-kit⁺ cells

(A) Representative FACS dot plots showing the distribution of CD45⁺/c-kit⁻, CD45⁻/c-kit⁺ and CD45⁺/c-kit⁺ cells from bone marrow, neonatal heart, and normal and infarcted adult heart. (B) Quantitative analysis of c-kit⁺ cells from bone marrow, neonatal heart and normal and infarcted adult heart. (C) Quantitative analysis of the percentage of c-kit⁺ cells co-expressing the hematopoietic marker CD45. Data represent mean ± SEM (n=6). *p < 0.05; n.s.: not significant.

Figure 2. Isolation of c-kit⁺ cells

c-kit⁺ cells were isolated using 2 consecutive isolation procedures with a MACS column. Representative cell preparations from neonatal (A) and normal adult (B) hearts are shown. The left panel shows EGFP epi-fluorescence, the middle panel shows c-kit⁺ immune reactivity (Rhodamine-conjugated secondary antibody) and the right panel shows Hoechst nuclear staining (blue). The insets depict high magnification images of a single cell from the field. Bar = 20 microns in low power images, 5 microns in insets.

Figure 3. Cardiac-resident c-kit⁺ cells from neonatal hearts exhibit robust cardiomyogenic differentiation when co-cultured with fetal cardiomyocytes

Non-transgenic fetal cardiomyocyte cultures were seeded with neonatal c-kit⁺ cells from ACT-EGFP/MHC-nLAC double transgenic hearts and cultured for 7 days. The cultures were then fixed and processed for β-galactosidase activity (blue), EGFP (green) and α-actinin (red) immune reactivity. EGFP-expressing cardiomyocytes exhibiting mature sarcomeric structure (yellow due to the overlay of green EGFP and red α-actinin signals) were readily detected by fluorescence microscopy; these cells also exhibited β-galactosidase activity when visualized under bright field illumination (inset). Bar = 20 microns in the fluorescent image, 20 microns in the bright field inset. Arrows mark β-galactosidase⁺ nuclei. Bright field, single color fluorescence and merged images for these cells are shown in the Supplemental Data section.

Figure 4. Cardiac-resident c-kit⁺ cells from neonatal hearts exhibit robust cardiomyogenic differentiation in the absence of cell fusion

Fetal cardiomyocyte cultures from single transgenic MHC-nLAC mice were seeded with c-kit⁺ cells from neonatal ACT-EGFP hearts and cultured for 7 days. The cultures were then fixed and processed for β-galactosidase activity (blue), EGFP (green) and α-actinin (red) immune reactivity. EGFP-expressing cardiomyocytes exhibiting mature sarcomeric structure (yellow due to the overlay of green EGFP and red α-actinin signals) were readily detected by fluorescence microscopy; however these cells lacked β-galactosidase activity when visualized under bright field illumination (inset). Bar = 20 microns in the fluorescent image, 20 microns in the bright field inset. Arrows mark β-galatosidase positive nuclei. Bright field, single color fluorescence and merged images for these cells are shown in the Supplemental Data section.

Figure 5. Cardiac-resident c-kit⁺ cells from normal adult hearts fail to undergo cardiomyogenic differentiation when co-cultured with fetal cardiomyocytes

Non-transgenic fetal cardiomyocyte cultures were seeded with c-kit⁺ cells from adult ACT-EGFP/MHC-nLAC double transgenic hearts and cultured for 7 days. The cultures were then fixed and processed for β-galactosidase activity (blue), EGFP (green) and α-actinin (red) immune reactivity. EGFP-expressing cells were readily detected; however these cells lacked sarcomeric structure when examined by fluorescence microscopy, and lacked β-galactosidase activity when visualized under bright field illumination (inset). Bar = 20 microns in the fluorescent image, 20 microns in the bright field inset. Bright field, single color fluorescence and merged images for these cells are shown in the Supplemental Data section.

Figure 6. Cardiac-resident c-kit⁺ cells from normal adult hearts fail to undergo cardiomyogenic differentiation when transplanted into infarcted adult mouse hearts

c-kit⁺ cells from ACT-EGFP/MHC-nLAC adult double transgenic hearts were transplanted into an infarcted non-transgenic recipient heart. The heart was harvested 13 days later, fixed and processed for β-galactosidase activity (blue), EGFP (green) and α-actinin (red) immune reactivity. EGFP-expressing cells were readily detected; however these cells lacked sarcomeric structure when examined by fluorescence microscopy and also lacked β-galactosidase activity when visualized under bright field illumination (inset). Bar = 20 microns in the fluorescent image, 20 microns in the bright field inset. Bright field, single color fluorescence and merged images for these cells are shown in the Supplemental Data section.

Figure 7. Low levels of apparent cardiomyogenic differentiation from cardiac-resident c-kit⁺ cells from infarcted adult hearts when co-cultured with fetal cardiomyocytes

Non-transgenic fetal cardiomyocyte cultures were seeded with c-kit⁺ cells from infarcted adult ACT-EGFP/MHC-nLAC double transgenic hearts and cultured for 7 days. The cultures were then fixed and processed for β-galactosidase activity (blue), EGFP (green) and α-actinin (red) immune reactivity. A single EGFP-expressing cardiomyocyte exhibiting mature sarcomeric structure (anti-EGFP immune reactivity, green, upper panel; anti-α-actinin immune reactivity, red, lower panel) was observed; this cell also exhibited β-galactosidase activity when visualized under bright field illumination (inset). Bar = 20 microns in the fluorescent image, 20 microns in the bright field inset. Arrows mark β-galactosidase⁺ nuclei. Bright field, single color fluorescence and merged images for these cells are shown in the Supplemental Data section.

Figure 8. The vast majority of cardiac-resident c-kit⁺ cells from infarcted adult hearts fail to undergo cardiomyogenic differentiation when co-cultured with fetal cardiomyocytes

Non-transgenic fetal cardiomyocyte cultures were seeded with c-kit⁺ cells from infarcted adult ACT-EGFP/MHC-nLAC double transgenic hearts and cultured for 7 days. The cultures were then fixed and processed for β-galactosidase activity (blue), EGFP (green) and α-actinin (red) immune reactivity. EGFP-expressing cells were readily detected; however these cells lacked sarcomeric structure when examined by fluorescence microscopy, and lacked β-galactosidase activity when visualized under bright field illumination (inset). Bar = 20 microns in the fluorescent image, 20 microns in the bright field inset. Bright field, single color fluorescence and merged images for these cells are shown in the Supplemental Data section.