Externally Applied Static Magnetic Field Enhances Cardiac Retention and Functional Benefit of Magnetically Iron-Labeled Adipose-Derived Stem Cells in Infarcted Hearts

Abstract

: Although adipose-derived stem cells (ASCs) hold the promise of effective therapy for myocardial infarction, low cardiac retention of implanted ASCs has hindered their therapeutic efficiency. We investigated whether an externally applied static magnetic field (SMF) enhances cardiac localization of "magnetic" cells and promotes heart function recovery when ASCs are preloaded with superparamagnetic iron oxide (SPIO) nanoparticles. The influence of SMF (0.1 Tesla) on the biological activities of SPIO-labeled ASCs (_SPIOASCs) was investigated first. Fifty-six female rats with myocardial infarction underwent intramyocardial injection of cell culture medium (CCM) or male _SPIOASCs with or without the subcutaneous implantable magnet (CCM-magnet or _SPIOASC-magnet). Four weeks later, endothelial differentiation, angiogenic cytokine secretion, angiogenesis, cardiomyocyte apoptosis, cell retention, and cardiac performance were examined. The 0.1-Tsela SMF did not adversely affect the viability, proliferation, angiogenic cytokine secretion, and DNA integrity of _SPIOASCs. The implanted _SPIOASCs could differentiate into endothelial cell, incorporate into newly formed vessels, and secrete multiple angiogenic cytokines. Four weeks after cell transplantation, the number of cardiac _SPIOASCs was significantly increased, vascular density was markedly enlarged, fewer apoptotic cardiomyocytes were present, and heart contractile function was substantially improved in the _SPIOASC-magnet treated rats in comparison with the _SPIOASC-treated rats. The _SPIOASCs could differentiate into endothelial cells, incorporate into vessels, promote angiogenesis, and inhibit ischemic cardiomyocyte apoptosis. An externally applied SMF offered a secure environment for biological properties of _SPIOASCs, increased the cardiac retention of implanted magnetic _SPIOASCs, and further enhanced heart function recovery after myocardial infarction.

Significance: This pilot proof-of-concept study suggests that a 0.1-Tesla static magnetic field does not adversely affect the viability, proliferation, angiogenic cytokine secretion, or DNA integrity of the superparamagnetic iron oxide-labeled adipose-derived stem cells (_SPIOASCs). Implantation of adipose-derived stem cells promotes myocardial neovascularization and inhibits ischemic cardiomyocyte apoptosis through endothelial differentiation, incorporation into vessels, and paracrine factor secretion. An externally applied static magnetic field enhanced myocardial retention of intramyocardially injected "magnetic" _SPIOASCs and promoted cardiac function recovery after myocardial infarction. With further preclinical optimization, this approach may improve the outcome of current stem cell therapy for ischemic myocardial infarction.

Keywords: Adipose-derived stem cells; Cell retention; Myocardial infarction; Static magnetic field; Superparamagnetic iron oxide.

Figure 1.

Characterization of ASCs. (A): The cultured ASCs (n = 3 experiments) were positive for CD29, CD59, and CD90.1 and negative for CD11b and CD45. (B): The ASCs appeared spindle-shaped under phase-contrast microscopy. (C): Fluorescence microscopy images of ASCs expressed strong blue GFP fluorescence. (D): Prussian blue staining showed blue magnetic SPIO particles distributed around the nuclei of _SPIOASCs. Scale bar = 50 μm. Abbreviations: ASC, adipose-derived stem cell; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; SPIO, superparamagnetic iron oxide; _SPIOASC, superparamagnetic iron oxide-labeled adipose-derived stem cell.

Figure 2.

Angiogenic cytokines secretion and DNA integrity of ASCs under the exposure of 0.1-Tesla static magnetic field. (A): Typical example of reverse-transcriptase polymerase chain reaction analysis of VEGF and IGF-1 expression in ASCs, _SPIOASCs, and _SPIOASCs-magnet. (B and C): Expression of two cytokines from three groups of ASCs were indistinguishable (n = 3 experiments for each cell group). (D): DNA damages with comet tail were not observed at three cell groups. (E): The percentage of cells with integral DNA was identical among three cell groups (n = 3 experiments for each cell group). Scale bar = 50 μm. Abbreviations: ASC, adipose-derived stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGF-1, insulin-like growth factor-1; _SPIOASC, superparamagnetic iron oxide-labeled adipose-derived stem cell; VEGF, vascular endothelial growth factor.

Figure 3.

Immunofluorescence analysis of the injected ASCs 4 weeks after cell transplantation. The sections of heart intramyocardially injected with GFP-positive ASCs (green) were costained with DAPI (blue) and vWF or three cytokines (red) in the _SPIOASCs treated rats (n = 8) and the _SPIOASC-magnet-treated rats (n = 8). (A): Merge images in right column showed that some implanted ASCs differentiated into endothelial cells. (B): Merge images in right column showed that some implanted ASCs directly incorporated into newly formed vessels. (C–E): Merge images in right column showed that the injected ASCs secreted VEGF (C), HGF (D), and IGF-1 (E) under myocardial microenvironment. Scale bar = 20 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; _SPIOASC, superparamagnetic iron oxide-labeled adipose-derived stem cell; vWF, von Willebrand factor.

Figure 3.

Immunofluorescence analysis of the injected ASCs 4 weeks after cell transplantation. The sections of heart intramyocardially injected with GFP-positive ASCs (green) were costained with DAPI (blue) and vWF or three cytokines (red) in the _SPIOASCs treated rats (n = 8) and the _SPIOASC-magnet-treated rats (n = 8). (A): Merge images in right column showed that some implanted ASCs differentiated into endothelial cells. (B): Merge images in right column showed that some implanted ASCs directly incorporated into newly formed vessels. (C–E): Merge images in right column showed that the injected ASCs secreted VEGF (C), HGF (D), and IGF-1 (E) under myocardial microenvironment. Scale bar = 20 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; _SPIOASC, superparamagnetic iron oxide-labeled adipose-derived stem cell; vWF, von Willebrand factor.

Figure 4.

Effect of the externally applied static magnetic field on cell retention and engraftment. (A, B): One week after cell implantation, more GFP-positive _SPIOASCs and cells containing blue-stained particles were detected in the _SPIOASC-magnet-treated rats (n = 6) than in the _SPIOASC-treated rats (n = 6). Regions 1 and 2 selected in left column are amplified in two right columns, respectively. (C): At 1 week after cell transplantation, the _SPIOASC-magnet-treated rats (n = 6) exhibited an approximately 1.86-fold greater cell numbers per high-power field than the _SPIOASC-treated rats (n = 6). (D): The reproducibility of the standard curve obtained by real-time polymerase chain reaction. A serial 10-fold dilution of DNA was tested 6 times in separate experiments. Each circle corresponds to the result of one dilution in one assay. The solid line corresponds to the regression analysis. (E, F): At 4 weeks after cell transplantation, the _SPIOASC-magnet treated rats (n = 6) showed an approximately 1.82-fold greater cell numbers per milligram of heart tissue (E) and per heart (F) than the _SPIOASC-treated rats (n = 6). Scale bar = 20 μm. ∗, p < .05 versus _SPIOASCs. Abbreviation: _SPIOASC, superparamagnetic iron oxide-labeled adipose-derived stem cell.

Figure 5.

Immunofluorescence analysis of capillary density in the peri-infarct zone 4 weeks after cell transplantation. (A): Cardiac sections from four rat groups were stained with DAPI (blue) and antibody to endothelial cell marker vWF (red). (B): Capillary density was greatest in the _SPIOADSC-magnet-treated rats (n = 8) compared with the _SPIOADSC-treated rats (n = 8), the CCM-magnet rats (n = 8), and the CCM control rats (n = 8). Scale bar = 50 μm. #, p > .05 versus the CCM; Δ, p < .05 versus the CCM or CCM-magnet; ∗, p < .05 versus the _SPIOASCs, CCM-magnet, or CCM. Abbreviations: CCM, cell culture medium; DAPI, 4′,6-diamidino-2-phenylindole; _SPIOASC, superparamagnetic iron oxide-labeled adipose-derived stem cell; vWF, von Willebrand factor.

Figure 6.

TUNEL staining of apoptotic cardiomyocytes in the peri-infarct zone 4 weeks after cell transplantation. (A): Cardiac sections from four rat groups were stained with DAPI (blue) and with TUNEL reagent for apoptosis (red). (B): TUNEL-positive cardiomyocytes were fewest in the _SPIOASC-magnet-treated rats (n = 8) compared with the _SPIOASC-treated rats (n = 8), the CCM-magnet rats (n = 8), and the CCM control rats (n = 8). Scale bar = 20 μm. #, p > .05 versus the CCM; Δ, p < .05 versus the CCM or CCM-magnet; ∗, p < .05 versus the _SPIOASCs, CCM-magnet, or CCM. Abbreviations: CCM, cell culture medium; DAPI, 4′,6-diamidino-2-phenylindole; _SPIOASC, superparamagnetic iron oxide-labeled adipose-derived stem cell; TUNEL, terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling.

Figure 7.

Magnetic resonance imaging (MRI) analysis of cardiac function. (A): Typical short-axis cine MRIs from end-diastole to end-systole during the whole cardiac cycle. The _SPIOASC-magnet-treated rats (n = 14) displayed a smaller left ventricular (LV) slice volume in comparison with the _SPIOASC-treated rats (n = 14), the CCM-magnet-treated rats (n = 8), and the CCM control rats (n = 8). (B, C): LV end-diastolic (B) and end-systolic (C) volumes were lowest in the _SPIOASC-magnet-treated rats (n = 14) compared with the _SPIOASC-treated rats (n = 14), the CCM-magnet rats (n = 8), and the CCM control rats (n = 8). (D): LV ejection fraction was highest in the _SPIOASC-magnet-treated rats (n = 14) compared with the _SPIOASC-treated rats (n = 14), the CCM-magnet rats (n = 8), and the CCM control rats (n = 8). #, p > .05 versus the CCM; Δ, p < .05 versus the CCM or CCM-magnet; ∗, p < .05 versus the _SPIOASCs, CCM-magnet, or CCM. Abbreviations: CCM, cell culture medium; _SPIOASC, superparamagnetic iron oxide-labeled adipose-derived stem cell.