Sustained subcutaneous delivery of secretome of human cardiac stem cells promotes cardiac repair following myocardial infarction

Abstract

Aims: To establish pre-clinical proof of concept that sustained subcutaneous delivery of the secretome of human cardiac stem cells (CSCs) can be achieved in vivo to produce significant cardioreparative outcomes in the setting of myocardial infarction.

Methods and results: Rats were subjected to permanent ligation of left anterior descending coronary artery and randomized to receive subcutaneous implantation of TheraCyte devices containing either culture media as control or 1 × 106 human W8B2+ CSCs, immediately following myocardial ischaemia. At 4 weeks following myocardial infarction, rats treated with W8B2+ CSCs encapsulated within the TheraCyte device showed preserved left ventricular ejection fraction. The preservation of cardiac function was accompanied by reduced fibrotic scar tissue, interstitial fibrosis, cardiomyocyte hypertrophy, as well as increased myocardial vascular density. Histological analysis of the TheraCyte devices harvested at 4 weeks post-implantation demonstrated survival of human W8B2+ CSCs within the devices, and the outer membrane was highly vascularized by host blood vessels. Using CSCs expressing plasma membrane reporters, extracellular vesicles of W8B2+ CSCs were found to be transferred to the heart and other organs at 4 weeks post-implantation. Furthermore, mass spectrometry-based proteomic profiling of extracellular vesicles of W8B2+ CSCs identified proteins implicated in inflammation, immunoregulation, cell survival, angiogenesis, as well as tissue remodelling and fibrosis that could mediate the cardioreparative effects of secretome of human W8B2+ CSCs.

Conclusions: Subcutaneous implantation of TheraCyte devices encapsulating human W8B2+ CSCs attenuated adverse cardiac remodelling and preserved cardiac function following myocardial infarction. The TheraCyte device can be employed to deliver stem cells in a minimally invasive manner for effective secretome-based cardiac therapy.

Keywords: Cardiac remodelling; Cardiac stem cells; Heart failure; Myocardial infarction; Secretome; TheraCyte.

Graphical abstract

Figure 1

Subcutaneous implantation of W8B2⁺ CSCs encapsulated in a TheraCyte device induces cardioprotection in a rat model of myocardial infarction. (A) Schematic of W8B2⁺ CSCs encapsulated in a TheraCyte device. (B) Representative M-mode view of echocardiography at 4 weeks following myocardial infarction and summarized mean data of ejection fraction (EF) (n = 6–7 independent experiments). (C) Representative images of left ventricular cross-sections stained with Masson’s trichrome and infarct size expressed as the percentage of fibrotic scar area over total left ventricle. (D) Representative images of collagen fibres (in red) stained with picrosirius red and percentage of interstitial fibrosis in the remote myocardium. (E) Representative images of left ventricular cross-sections stained with wheat germ agglutinin and the relative cross-sectional area of cardiomyocytes. (F) Total vascular density determined in lectin-stained sections. (G) Arteriole density determined in smooth muscle actin-stained sections. n = 7–8 independent experiments. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control by unpaired Student’s t-test or by two-way ANOVA with Bonferroni post hoc test in (B).

Figure 2

Encapsulated W8B2⁺ CSCs in TheraCyte devices. (A, B) Eosin–haematoxylin stained Theracyte sections at 0 (A) and 4 (B) weeks post-implantation. (C–E) TheraCyte devices with encapsulated W8B2⁺ CSCs at 4 weeks post-implantation and stained with human-specific KU80 antibody (C), lectin (D, arrows indicate erythrocytes), cleaved caspase-3 antibody (E) and Ki67 antibody (F). (E) The percentage of cell area within the TheraCyte stained positive for cleaved caspase-3 (arrows) at Day 28 post-implantation. n = 7. (F) The percentage of Ki67 positive cells (arrows) encapsulated within the TheraCyte devices at Day 28 post-implantation. n = 7 independent experiments.

Figure 3

Characterization of extracellular vesicles secreted by encapsulated W8B2⁺ CSCs. (A) Transduced W8B2+ cells expressing both the GlucB-IRES-GFP and sshBirA-IRES-mCherry vectors (W8B2⁺ CSC^{GlucB+sshBirA}), resulting in extracellular vesicles with GlucB and sshBirA labelled and biotinylated on the surface of the plasma membrane. (B) W8B2⁺ CSC^{GlucB+sshBirA} encapsulated within a TheraCyte device and expressing mCherry and GFP fluorescence proteins in vitro. (C, D) Gluc activity (C) and concentration of microvesicles (D) in conditioned media (50 µL and centrifuged at 500 g) harvested at 1, 2, and 3 days from W8B2⁺ CSC^{GlucB+sshBirA} cultured as monolayer (2D) or encapsulated within a TheraCyte device (3D). n = 3 independent experiments. Data are shown as mean ± SEM. *P < 0.05, ****P < 0.0001 by one-way ANOVA with Bonferroni post hoc test. (E, F) Nanoparticle tracking analysis showing the size distribution of EVs in conditioned media harvested at 1, 2, and 3 days from W8B2⁺ CSC^{GlucB+sshBirA} cultured as monolayer (E) or encapsulated within a TheraCyte device (F). (G) Size distribution of EVs in conditioned media harvested on Day 3 (∼1.5 mL and centrifuged at 2000 g followed by 110 000 g twice) from untransduced W8B2⁺ CSCs and W8B2⁺ CSC^{GlucB+sshBirA} encapsulated within a TheraCyte device. n = 3 independent experiments.

Figure 4

Characterization of extracellular vesicles secreted by W8B2⁺ CSCs. (A) PCA analysis of proteins identified in untransduced cells (UnT Cell), matched EVs (UnT EV), and transduced EVs (T EV). Unsupervised PCA analysis was conducted using the average protein LFQ intensities of technical replicates. Correlation matrix was visualized with illustrated Pearson correlation coefficient using psych package in R. (B) Volcano plot, statistically significant proteins with less than 0.05 adjusted P-value and greater than 1.5 log2 fold change differences. (C) Exosome marker proteins identified and enriched in UnT EV and T EV relative to UnT cells. (D) Distribution of protein identified between UnT EV and T EV. (E) Multi-variate analysis of intra- (technical) and inter (biological) sample variation of UnT EV and T EV. (F–G) KEGG functional enrichment analysis of UnT EV, reveals significant enrichment, relative to human genome (P < 0.01), of the ROBO receptor signalling (HSA-376176) (F) and NOTCH signalling (HSA-157118) (G).

Figure 5

Biodistribution of extracellular vesicles secreted by encapsulated W8B2⁺ CSCs. (A, B) W8B2⁺ CSC^{GlucB+sshBirA} encapsulated within a TheraCyte device were implanted subcutaneously into rats following myocardial infarction. At 4 weeks post-implantation, paraffin-embedded sections were stained with horseradish peroxidase-conjugated streptavidin to detect biotin (A) or with GFP antibody (B) showing positive punctate staining across and outside the inner membrane and in cells within the vicinity of the TheraCyte device (arrows). (C, D) Cryosections of the myocardium showed retention of extracellular vesicles of W8B2⁺ CSC^{GlucB+sshBirA} (arrows) in the interstitial space and inside the cardiomyocytes (C), as well as in the perivascular region (D). The super-resolution confocal images are the enlarged views of the field covered by the white boxes shown in the confocal images.