Cardiosphere-derived cells improve function in the infarcted rat heart for at least 16 weeks--an MRI study

Abstract

Aims: Endogenous cardiac progenitor cells, expanded from explants via cardiosphere formation, present a promising cell source to prevent heart failure following myocardial infarction. Here we used cine-magnetic resonance imaging (MRI) to track administered cardiosphere-derived cells (CDCs) and to measure changes in cardiac function over four months in the infarcted rat heart.

Methods and results: CDCs, cultured from neonatal rat heart, comprised a heterogeneous population including cells expressing the mesenchymal markers CD90 and CD105, the stem cell marker c-kit and the pluripotency markers Sox2, Oct3/4 and Klf-4. CDCs (2 × 10(6)) expressing green fluorescent protein (GFP+) were labelled with fluorescent micron-sized particles of iron oxide (MPIO). Labelled cells were administered to the infarcted rat hearts (n = 7) by intramyocardial injection immediately following reperfusion, then by systemic infusion (4 × 10(6)) 2 days later. A control group (n = 7) was administered cell medium. MR hypointensities caused by the MPIOs were detected at all times and GFP+ cells containing MPIO particles were identified in tissue slices at 16 weeks. At two days after infarction, cardiac function was similar between groups. By 6 weeks, ejection fractions in control hearts had significantly decreased (47 ± 2%), but this was not evident in CDC-treated hearts (56 ± 3%). The significantly higher ejection fractions in the CDC-treated group were maintained for a further 10 weeks. In addition, CDC-treated rat hearts had significantly increased capillary density in the peri-infarct region and lower infarct sizes. MPIO-labelled cells also expressed cardiac troponin I, von Willebrand factor and smooth muscle actin, suggesting their differentiation along the cardiomyocyte lineage and the formation of new blood vessels.

Conclusions: CDCs were retained in the infarcted rat heart for 16 weeks and improved cardiac function.

Figure 1. Characterisation of CDCs.

Surface marker expression and mRNA in passage 2 CDCs was measured using (A, B) flow cytometry for c-kit, CD90 and DDR2, representative plots are shown in (A) and quantication in (B); (C) qRT-PCR for c-kit, GATA-4, CD105 and CD90, expressed relative to 100% expression in neonatal heart and normalised to GAPDH; and (D) immunocytochemistry for Oct3/4, Sox2, Nanog, KLF-4, c-kit, GATA-4, Nkx2.5, von Willibrand factor (vWF), α–smooth muscle actin (α–SMA), α–sarcomeric actin and Ki67. (E & F) Incubation with DMSO for 10 days promoted differentiation along the cardiac lineage as shown by increased protein expression of cTroponin I (E: green) and mRNA expression of Nkx2.5, Troponin T and myosin heavy chain (MyHC) (F).

Figure 2. CDCs were not adversely affected by labelling with MPIOs and could be detected using MR microscopy.

(A) CDCs endocytosed fluorescent MPIOs during overnight incubation; (B) MPIO-labelled CDCs retained the ability to form cardiospheres; (C) live (blue) and dead (red) cell numbers were comparable between unlabelled and MPIO-labelled CDCs; (D) MPIO-labelled CDCs could be detected at the single cell level (10³ cells / 0.5 µl, arrows) using MR microscopy.

Figure 3. CDC administration improved cardiac function.

Cardiac ejection fraction, end systolic volume, end diastolic volume, stroke volume, relative infarct size and end-systolic peri-infarct thickness, measured over 16 weeks using MRI, showed that cardiac function in the infarcted hearts was improved by administration of CDCs. * p<0.05 vs sham ; # p<0.05 vs control; † p<0.05 vs 2 days (shown only for infarct size and peri-infarct thickness for clarity).

Figure 4. MPIO-labelled CDCs were tracked in vivo and identified in tissue sections.

A: Regions of hypointensity due to MPIOs (arrows) were detected in CDC-treated hearts using in vivo MRI for 16 weeks after administration, and confirmed using ex vivo MR microscopy (example images from the same heart at 2 days, 2 weeks and 16 weeks are shown, in vivo top, left & right, bottom left, ex vivo bottom right). B: In tissue slices, MPIOs (green) were shown to co-localise with expression of GFP (magenta). C: Macrophages were detected in tissue slices, by antibody labelling for CD68 (magenta, yellow arrowheads), but were not labelled with DiI (red) or MPIOs (green, white arrows). D: No vimentin positive/MPIO/DiI positive cells were observed and therefore the cells did not appear to be myofibroblasts.

Figure 5. Differentiating CDCs were detected in tissue sections.

Regenerating myocardial cells were detected by co-localisation of MPIO (green) and DiI (red) labelling with (A) immuno-staining for cTnI (magenta); (B) vWF-positive capillaries (magenta) and (C) SMA-positive arterioles (magenta, inset is at ×2 zoom), indicating de-novo cardiomyocyte and vessel formation from administered CDCs. (D) MPIO/cTnI positive small immature cells within the border zone expressed a small amount of connexin 43 (arrow), but these cells did not form gap junctions with mature cardiomyocytes.

Figure 6. CDC administration reduced infarct size and increased capillary density.

Infarct size measurements obtained using picro-Sirius red staining (A: top) and MRI (A: bottom) were found to correlate (B) and showed that CDC-treated hearts were less fibrotic than control hearts (C). Immuno-staining for CD31 showed increased capillary density in the peri-infarct region of CDC-treated hearts compared with controls; representative images are shown in (D) and quantified in (E).