Cardiac recovery via extended cell-free delivery of extracellular vesicles secreted by cardiomyocytes derived from induced pluripotent stem cells

Abstract

The ability of extracellular vesicles (EVs) to regulate a broad range of cellular processes has recently been exploited for the treatment of diseases. For example, EVs secreted by stem cells injected into infarcted hearts can induce recovery through the delivery of stem-cell-specific miRNAs. However, the retention of the EVs and the therapeutic effects are short-lived. Here, we show that an engineered hydrogel patch capable of slowly releasing EVs secreted from cardiomyocytes derived from induced pluripotent stem (iPS) cells reduced arrhythmic burden, promoted ejection-fraction recovery, decreased cardiomyocyte apoptosis 24 hours after infarction, and reduced infarct size and cell hypertrophy 4 weeks post-infarction when implanted onto infarcted rat hearts. We also show that the EVs are enriched with cardiac-specific miRNAs known to modulate cardiomyocyte-specific processes. The extended delivery of EVs secreted from iPS-cell-derived cardiomyocytes into the heart may help understand heart recovery and treat heart injury.

Figure 1. Therapeutic potential and challenges of cell and extracellular vesicle-based therapies

Cell based therapies utilizing either stem cells or differentiated cardiomyocytes have shown clinical utility in their ability to re-functionalize the injured heart. When directly injected into the heart, both stem cells and cardiomyocytes have resulted in the re-functionalization of the heart and improved clinical outcomes^–. However, many challenges arise from the use of cell therapies. Specifically, stem cell therapies only demonstrate modest effects while cardiomyocyte therapies show high propensity of arrhythmogenicity^,,. Stem cell extracellular vesicles (EVs) have also been able to re-functionalize the injured heart, mediating the regeneration of the heart after myocardial infarction^,,,. However, the cargo of stem cell EVs is not specific to cardiac processes. We hypothesize that iCMs, unlike naïve iPS cells, secrete EVs carrying cardiomyocyte specific cargo that can target the myocardium, providing protection from injury and promoting recovery after myocardial infarction.

Figure 2. iCMs secrete functional EVs

a) Representative size distribution of microvesicles isolated from iPS cells and iCMs. b) Total number and size of microvesicles (Average ± SEM, n = 4 and 8 biologically independent samples for iPS and iCM groups respectively). c) Representative transmission electron micrographs of microvesicles (scale = 100 nm, experiments were repeated with similar results). d) Immunoblots of cell lysates and microvesicle fractions for the exosome marker TSG101, and intracellular proteins (GM130, Rab5) (n = 3 biologically independent samples per group). e) Representative bioanalyzer plots of total RNA profiles of iCMs and iCM-EVs (n = 3 biologically independent samples per group). f) Representative traces of spontaneously beating iCMs at baseline (normoxia) and after 48 hours of hypoxia, without EVs, and with iPS or iCM-EVs (experiments were repeated with similar results). Range (g) and standard deviation (h) of the distributions of instantaneous beating frequencies. Boxes show mean, 25th and 75th percentiles. Whiskers show 9th to 91st percentile. (n = 49, 35, 12, and 31 biologically independent samples for baseline, PBS, iPS-Ev, and iCM-Ev groups respectively, * denotes p < 0.05 by two sided T-test).

Figure 3. iCM-EVs are enriched in cardiac specific miRNAs

a) Principal component analysis of iPS-EV and iCM-EV miRNA expression (n = 4 biologically independent samples per group). b) Fold change and expression levels (Tags per million) of the most abundant miRNAs in iCM-EVs that are differentially expressed. c) Volcano plot of significantly differentially expressed miRNAs. P-values calculated via Wald test and adjusted with the Benjamini-Hochberg method (n = 4 biologically independent samples per group). d) Differentially expressed and abundantly expressed miRNAs in iCM-EVs. Highlighted region contains miRNAs used in subsequent gene ontology analysis. (n = 4 biologically independent samples per group). Fold change over expected enrichment shown in parenthesis; cardiac related processes highlighted in gray.

Figure 4. Hydrogel patch sustainably released encapsulated EVs in a rat heart infarction model

a) Cumulative release profile of EV-containing patches over 21 days in vitro as quantified by Nanosight (n = 2 biologically independent samples). b) Representative image of DiI stained EVs present in hydrogel patches explanted 0, 4, and 7 days after implant onto rat myocardium. (n = 1 biologically independent sample per day) (Scale bar 200uM) c) Schematic representation of the experimental set-up for the rat model: EVs are isolated from iPS cells or iCMs and incorporated into a patch as therapy in a rat acute myocardial infarction model.

Figure 5. iCM-EVs are non-arrhythmogenic and promote recovery of heart contractile function

a) Electrocardiogram progression after LAD. All rats exhibited ST-elevations following LAD ligation, followed by Q waves and T-wave inversion within 3 days. LAD ligation resulted in arrhythmic events including atrioventricular block, characterized by a p-wave without a subsequent QRS complex, premature ventricular contraction, characterized by a widened QRS complex without a preceding p-wave, ventricular tachycardia, characterized by more than three consecutive QRS complexes without preceding p-waves, and ventricular fibrillation, characterized by uncoordinated electrical activity. b) Number of arrhythmic events per hour experienced by animals during the first 5 days after infarction (Average ± SEM, n = 3, 5, 5, 7, and 6 biologically independent samples for sham, MI-only, PBS-P, iPSev-P, and iCMev-P groups respectively)(* denotes p<0.05 by two sided T-test). c) Number of arrhythmic events per hour experienced by animals for 3 hours after isoproterenol challenge (Average ± SEM, n = 3, 5, 5, 7, and 5 biologically independent samples for sham, MI-only, PBS-P, iPSev-P, and iCMev-P groups respectively)(* denotes p<0.05 by two sided T-test). d) Quantified echocardiogram parameters of animals at 2 and 4-week time points. (Average ± SEM, n = 5, 4, 6, 7, 6 biologically independent samples for sham, MI-only, PBS-P, iPSev-P, and iCMev-P groups respectively). e) Statistical significance of quantified echocardiogram parameters between all groups at 4 weeks quantified by two tailed T-test. Red indicates a significant difference with Bonferroni corrected p < 0.05. f) Representative M-mode echocardiographs at 4 weeks (experiments were repeated in n = 6, 5, 6, 7, and 6 animals for sham, MI-only, PBS-P, iPSev-P, and iCMev-P groups respectively with similar results).

Figure 6. iCM-EV treatment reduced infarct size and CM hypertrophy

a) Representative transverse cardiac sections stained with Movat’s pentachrome stain (scale bars = 1 mm). The letter P indicates the location of the patch. b) High power image of the infarct border zone (scale bars = 100 μm) (experiments were repeated in n = 5, 5, 7, 6 biologically independent samples for MI-only, PBS-P, iPSev-P, and iCMev-P groups respectively with similar results). c) Infarct size as a percentage of the total left ventricle (Average ± SEM, n = 5, 5, 7, 6 biologically independent samples for MI-only, PBS-P, iPSev-P, and iCMev-P groups respectively, * denotes p < 0.05 by two-tailed T-test). d) Sections were stained for wheat germ agglutinin (red), troponin (green), and DAPI (blue), and e) relative cardiomyocyte area was quantified. Scale bars = 50 μm. (Average ± SEM, n = 6, 5, 6, 6, and 6 biologically independent samples for sham, MI-only, PBS-P, iPSev-P, and iCMev-P groups respectively with similar results, * denotes p < 0.05 by two-tailed T-test).

Figure 7. iCM-EV treatment prevents apoptosis in the acutely infarcted heart

a) Quantification of ejection fraction 24 hours after LAD ligation (Average± SEM, n = 9, 10, and 8 biologically independent samples for MI-only, iPSev-P, iCMev-P groups respectively, * denotes p < 0.05 by two-tailed T-test) b) Activated caspase 3 (green), troponin (red), and DAPI (blue) stained cardiomyocytes treated with iCM-EVs or PBS and subjected to hypoxia (scale bars = 100 um) c) Quantification of the numbers of caspase 3 positive hypoxic cardiomyocytes relative to normoxic cardiomyocytes (F.C. denotes fold change, Average± SEM, n = 7 biologically independent samples per group, * denotes p <0.05 by two-tailed T-test). d) Representative transverse cardiac sections stained with TUNEL (scale = 1 mm) at high magnification (scale bars = 100um) e) Quantification of apoptotic area as a fraction of total left ventricular area. (Average ± SEM, n = 4 and 3 biologically independent samples for MI-only and iCMev-P groups respectively, * denotes p<0.05 by two-tailed T-test).