Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors

Abstract

Aims: We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act.

Methods and results: The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left ventricular end diastolic volume: -4%, P = 0.002), and increased LVEF (+14%, P < 0.0001) relative to baseline values. Gene profiling revealed that EV-treated hearts were enriched for tissue reparative pathways.

Conclusion: Extracellular vesicles secreted by iPSC-Pg are effective in the treatment of CHF, possibly, in part, through their specific miRNA signature and the associated stimulation of distinct cardioprotective pathways. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation.

Figures

Figure 1

Characterization of induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicle preparations. (A) Similar overall concentrations and yields of particles per cell were found in three separate preparations of induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicles by Nanoparticle Tracking Analysis. Cryo-transmission electron microscopy of these extracellular vesicles identified membrane-bound vesicles of the size of (B) exosomes, (C) microparticles, and (D) large vesicular bodies that may be apoptotic bodies.

Figure 2

Induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicles improve cardiac cell survival of immortalized H9c2 rat cardiac myoblasts. At T0, media was refreshed with complete media (positive control, dark grey bar), stressful serum free media (Dulbecco's Modified Eagle Medium glutamax, +1% penicillin/streptomycin/Amphotericin B) (negative control, bright grey bar), or serum free media plus 5.3 × 109 foetal bovine serum-derived extracellular vesicles (medium grey bar) or induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicles at Day 4 of culture at increasing concentrations (blue bars). At T = 27 h, the number of viable cells from each condition was determined and normalized to the number of cells at the start of the stress (T0 = 100%). Results are the average of 1–3 independent experiments, each in duplicate. Extracellular vesicle concentrations were determined by Nanoparticle Tracking Analysis at high camera sensitivity (camera level 15).

Figure 3

Induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicles exhibit pro-angiogenic effects in a scratch wound assay. The potential for induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicles to affect human umbilical vein endothelial cell migration was evaluated by the scratch wound assay. A scratch was induced in a human umbilical vein endothelial cell monolayer. (A) The percent wound invasion was determined at numerous time points, with the 18-h time point showing clear and consistent differences between (B) positive control (complete media = endothelial cell growth medium plus endothelial cell growth medium supplement pack; dark grey bar), and (C) negative control (poor media = endothelial cell growth medium; bright grey bar) with 97% and 10% healing, respectively. (D) Human umbilical vein endothelial cell invasion was increased by extracellular vesicle incubation (poor media + extracellular vesicles), even at the lowest concentration tested, with a seemingly dose-dependent increase in cell migration, reaching 80% invasion with 1 × 1010 extracellular vesicles per 15 000 human umbilical vein endothelial cells.

Figure 4

Induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicles stimulate human embryonic stem cell derived-cardiomyocyte proliferation. EdU incorporation was used to assess the potential for extracellular vesicles to stimulate cardiomyocyte proliferation. After 48 h of extracellular vesicle incubation, human embryonic stem cell derived-cardiomyocytes were incubated with EdU for an additional 24 h. (A) An α-actinin staining confirmed the cell phenotype. (B) The basal rate of proliferative cells was 15% which increased in a seemingly dose-dependent manner up to 35% of cells at 1.81 × 109 extracellular vesicles per 10 000 human embryonic stem cell derived-cardiomyocyte.

Figure 5

Induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicle treatment significantly improves cardiac function. Left ventricular end systolic volume, left ventricular end diastolic volume, and ejection fraction were determined 3 weeks after myocardial infarction (baseline) and 7 weeks after treatment. All groups showed some decrease in ventricular volumes and increase in ejection fraction. For the extracellular vesicle group, there is a clear and highly significant improvement in all three functional parameters, representing a decrease in (A) left ventricular end systolic volume and (B) left ventricular end diastolic volume of 11% and 4% relative to baseline, respectively, and an increase in (C) ejection fraction of 14% relative to baseline. (D) Considering left ventricular end systolic volume changes of individual mice in each group, significantly more mice responded to treatment (>5% decrease, ‘responders’, below the line) in the extracellular vesicle group than in any other group (P = 0.04, χ2 test). Analysis of variance indicates that the evolution of individual mice in the extracellular vesicle group is significantly more homogeneous than the evolution of mice in the induced pluripotent stem cell-derived cardiomyocytes and progenitor cell groups.

Figure 6

Induced pluripotent stem cell-derived cardiomyocytes, induced pluripotent stem cell-derived cardiovascular progenitors, and induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicle treatments lead to distinct gene expression patterns 7 weeks after treatment. Gene expression patterns in mouse hearts 7 weeks after treatment were analysed via mRNA array. Genes differentially expressed in each group when compared with the control phosphate-buffered saline group were identified and gene lists were compared with one another to identify common and specific differentially expressed genes. One-hundred and fifty-eight genes were common to all three treatments, whereas induced pluripotent stem cell-derived cardiomyocytes, induced pluripotent stem cell-derived cardiovascular progenitors, and induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicles differentially expressed 350, 1090, and 882 genes, respectively.

Figure 7

Heat map of differential functions by group—specific gene lists. Differentially expressed genes from each treatment group were assessed by Ingenuity to predict upregulated (warm colours) and downregulated (cool colours) diseases and biological functions when compared with phosphate-buffered saline controls. Each column examines differentially expressed genes uniquely present in the indicated treatment group (left—induced pluripotent stem cell-derived cardiomyocytes; middle—induced pluripotent stem cell-derived cardiovascular progenitors; right—induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicles). Induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicle treated hearts were the most enriched for functions consistent with increased cell proliferation/growth, cardiogenesis, and vessel formation. The list is sorted to have the greatest difference in enrichment between the extracellular vesicle group and the other groups at the top of the list. This list was truncated after the 78th line.

Take home figure

Human induced pluripotent stem cell-derived cardiovascular progenitor cell ‘biofactories’ produce therapeutic extracellular vesicles that benefit cells and tissues, improving outcomes. Human induced pluripotent stem cells are 1-expanded and 2-differentiated to cardiovascular progenitors. Under the appropriate culture conditions, the latter release extracellular vesicles which can be 3-concentrated and purified, eliminating the secretory cells. The human induced pluripotent stem cell-derived cardiovascular progenitor cell–extracellular vesicles contain a unique miRNA signature, including miRNAs associated with cardio protective and reparative pathways. In vitro, 4-these extracellular vesicles are internalized by target cells leading to cellular effects consistent with cardio-protection or repair. When 5-extracellular vesicles are delivered to the infarcted heart tissue of mice in chronic heart failure, human induced pluripotent stem cell-derived cardiovascular progenitor–extracellular vesicles appear to stimulate transcriptomic changes leading to improved cardiac function.