Complementary Embryonic and Adult Cell Populations Enhance Myocardial Repair in Rat Myocardial Injury Model

Figures

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  Figure 1. Characterization of isolated embryonic cardiac stem cells in culture. (a)–(c) Immunofluorescence micrographs showing cells isolated from E12.5 rat hearts expressing various cardiac progenitor markers: Isl-1 (a), Nkx2.5 (b), and GATA-4 (c). (d) Blank, in which the primary antibody was omitted. (e) Bright field and superimposed fluorescence micrograph of cells stained with Prussian Blue demonstrate incorporation of MION in the cytoplasm A close-up view of (e) is shown (e'). Light blue: nuclei (DAPI); dark blue: MION. (f) Western blot analysis of cell extract confirmed expression of various cardiac markers (Islet-1, Nkx2.5, GATA-4, sarcomeric α-actinin, cardiac troponin I, connexin-43). Cells were identified as c-kit−.(g) Real-time PCR analysis showed that, after several passages in culture, isolated cells lost expression of GATA-4.

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  Figure 2. Biodistribution of injected cells using MION labeling and FISH, showing that cell injections do not increase vascular density: (a) Isl-1+ cells retain MION labeling, as shown by Prussian blue staining, 4 weeks following injection with cells infiltrating the scar tissue (between yellow dotted lines) suggesting participation in the repair process. (b) Male donor-derived CD90+ cells were detected 4-week post-MI by fluorescence in situ hybridization (FISH) for the Y chromosome shown in red (arrows). (c)–(g) Representative images from all cohorts of cardiac tissue sections stained for von Willebrand factor. (c) sham; (d) saline; (e) Isl-1+; (f) CD90+; (g) Isl-1+/CD90+). Scale bar: 200 μm. (h) Quantification of all the sections analyzed (saline: 0.0388 μm2 ± 0.008; sham: 0.0340 μm2 ± 0.002; Isl-1: 0.0737 μm2 ± 0.02; CD90+: 0.02 μm2 ± 0.005; Isl-1+/CD90+: 0.025 μm2 ± 0.01).

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  Figure 3. Combination of Isl-1+ cells/CD90+ cells preserves wall thickness. (a) Masson's trichrome staining was used to stain the fibrotic tissue in blue. Representative bright field microscopy images of 4-week post-MI rat hearts are shown. Fibrosis is limited to the anterior wall of the left ventricle. (b) Quantification of fibrosis revealed that none of the cell treatments was able to significantly reduce fibrosis (saline: 13.85% ± 0.96 vs. Isl-1+: 10.69% ± 1.77, p = 0.14; saline vs. CD90+: 11.95% ± 1.61, p = 0.22; saline vs. Isl-1+/CD90+: 11.62% ± 1.55, p = 0.19). The combination of Isl-1+cells and CD90+ cell treatment was sufficient to maintain wall thickness (sham: 2354.39 μm ± 18.03 vs. saline: 996.98 μm ± 120.41, p < 0.05; sham vs. Isl-1+: 834.91 μm ± 54.50, p < 0.05; sham vs. CD90+: 1711.40 μm ± 16.67, p < 0.05; sham vs. Isl-1+/CD90+: 1841.23 μm ± 228.08, p = 0.02) (c).

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  Figure 4. Combination of Isl-1+cells and CD90+ cells leads to improved cardiac function, and schematic representation of combination stem cell administration on cardiac repair following LAD ligation induced MI. (a) Representative screenshots of echocardiograms at 1- and 4-week post-MI time points in all the four treatment groups (saline, Isl-1+, CD90+, and Isl-1+/CD90+). (b) Hemodynamic parameter assessment by Millar catheterization revealed that the combination therapy group demonstrated a significant improvement of cardiac function compared to either monotherapy. There was no significant difference between Isl-1+/CD90+ and sham. (c) Isl-1+ cells derived from mouse embryos and adult bone marrow-derived CD-90+ cells work in combination to restore cardiac function and preserve wall thickness in the LAD ligation model of MI. In vivo cells work in synergy to carry out healing and the use of two cell types may serve to optimize outcomes both for the target tissue in need of repair and for facilitating donor cell survival.