Pretreatment of Cardiac Stem Cells With Exosomes Derived From Mesenchymal Stem Cells Enhances Myocardial Repair

Abstract

Background: Exosomes derived from mesenchymal stem cells (MSCs) were proved to boost cell proliferation and angiogenic potency. We explored whether cardiac stem cells (CSCs) preconditioned with MSC exosomes could survive and function better in a myocardial infarction model.

Methods and results: DiI-labeled exosomes were internalized with CSCs. They stimulated proliferation, migration, and angiotube formation of CSCs in a dose-dependent manner. In a rat myocardial infarction model, MSC exosome-preconditioned CSCs had significantly better survival, enhanced capillary density, reduced cardiac fibrosis, and restored long-term cardiac function. MicroRNA profiling analysis revealed that a set of microRNAs were significantly changed in CSCs after MSC exosome treatment.

Conclusions: Pretreatment of CSCs with MSC exosomes provided a promising strategy to improve survival and angiogenic potency of CSCs.

Keywords: angiogenesis; cardiac stem cells; exosomes; miRNA profiling; proliferation.

Figure 1

Characterization of rat bone marrow mesenchymal stem cells (MSCs). A, The morphology of MSCs was observed under microscope. B, Representative fluorescence‐activated cell sorting (FACS) analysis of MSCs expressing antigens for CD90, CD34, CD44, and CD29. FS, fractional shortening.

Figure 2

Identification of mesenchymal stem cell (MSC) exosomes (MSC‐Exo). A, The morphology of MSC‐Exo was observed under an electron microscope. Bar, 200 nm. B, The diameter distribution of MSC‐Exo was measured by Image‐Pro Plus software. C, The phenotype of MSC‐Exo for CD63 was identified by fluorescence activated cell sorting (FACS) analysis. D, Western blot analysis of CD63 protein was detected in MSC‐Exo.

Figure 3

Identification of c‐kit⁺ cardiac stem cells (CSCs). The percentages of c‐kit for CSCs were performed by FACS analyses before (A) and after (B) Magnetic‐activated cell sortin (MACS). The morphology of c‐kit⁺ CSCs was observed under light microscopy. Bar, 250 μm, (C). Immunofluorescence for c‐kit was observed in the MACS‐sorted CSCs. Bar, 200 μm, (D). The differentiation of c‐kit⁺ CSCs was detected by immunofluorescence for cardiomyocyte specific protein (desmin, connexin‐43, smooth muscle specific protein [α‐SMA]), bar, 100 μm (E), and endothelial specific protein (CD31), bar, 20 μm (F). The proliferation of c‐kit⁺ CSCs was observed for immunofluorescent Ki‐67. Bar, 20 μm (G).

Figure 4

Cellular internalization of mesenchymal stem cell (MSC)‐Exo into c‐kit⁺ cardiac stem cells (CSCs). DiI‐labeled MSCs‐Exo (red) were internalized into DAPI‐labeled CSCs (blue). Bar, 25 μm.

Figure 5

Mesenchymal stem cells (MSCs)‐Exo enhanced in vitro proliferation, migration, and tube formation of c‐kit⁺ cardiac stem cells (CSCs) in a dose‐dependent manner. The c‐kit⁺ CSCs were incubated in the presence of different concentrations of MSCs‐Exo. A, After culture for 72 hours, proliferative capacity of CSCs was significantly improved dose‐dependently using CCK8 assay. B, Cell migration capacity was tested by transwell assay. The representative images were shown and the number of migrating cells per high‐power field was provided in different groups. Bar, 100 μm. C, Numbers of migrated cells were analyzed by Prism 5 software (n=5). D, Tube formation of CSCs in vitro in Matrigel. The representative images of capillary‐like structure were shown, and the length of tube per high field in different groups was measured by Image J software. Bar, 100 μm. E, Statistical analysis of tube length (n=5). The P values for comparison were indicated in the images, respectively.

Figure 6

Differentiation of transplanted cardiac stem cells (CSCs) into the neovasculature in the peri‐infarct myocardium. CSCs were transduced with lentiviral vector–green fluorescent protein (GFP), to be stably traced in vivo. CSCs^Exo were preconditioned with 400 μg/mL mesenchymal stem cells (MSCs)‐Exo for 24 hours, and CSCs were injected into the peri‐infarct zones 2 days after myocardial infarction induction. Heart samples were harvested following BS1 lectin systemic perfusion 28 days after cell injection. A, The transplanted CSCs that had differentiated into endothelial cells were visualized by immunofluorescence costaining for GFP (green) and BS1 lectin (red), in both groups. Bar, 25 μm. B, The numbers of transplanted cells that colocalized with capillaries were counted under a fluorescence microscope separately and averaged. CSCs^Exo vs CSCs, P<0.05; n=4.

Figure 7

Histological analysis for capillary density in the peri‐infarct myocardium. Cardiac stem cells (CSCs)^Exo were preconditioned with 400 μg/mL mesenchymal stem cells (MSCs)‐Exo for 24 hours), CSCs, and PBS control were injected into the peri‐infarct zones 2 days after myocardial infarction induction. Heart samples were harvested following BS1 lectin systemic perfusion 28 days after cell transplantation. A, Capillaries were immunofluorescently observed as tubular structure perfused with BS1 lectin (red). Bar, 100 μm. B, The numbers of capillaries were counted in the peri‐infarct zone and averaged in each group (n=5). C, Hearts sections were stained with anti–α–smooth muscle actin (SMA) antibody (red) to detect arterioles in the peri‐infarct zone. Bar, 100 μm. D, The numbers of arterioles were counted and averaged in each group (n=4).

Figure 8

Histological analysis for myocardial infarction (MI) sizes in each group. Cardiac stem cells (CSCs)^Exo were preconditioned with 400 μg/mL mesenchymal stem cells (MSCs) for 24 hours, and CSCs were injected into the peri‐infarct zones after MI induction. Heart samples were harvested 28 days after cell injection. A, Heart sections were stained with Masson trichrome: myocardium (red), scarred fibrosis (blue). The percentage of fibrotic area (B) and fibrosis length (C) was calculated and averaged (n=5) by using Image J software.

Figure 9

Functional analysis of rat hearts by echocardiography. A, Left ventricular ejection fraction (LVEF) was measured in the control, cardiac stem cells (CSCs). and CSCs^Exo‐injected groups. B, The changing tendency of LVEF preoperatively and 7 days and 28 days postoperatively was indicated (n=5). *Control vs CSCs and CSCs^Exo at postoperative day 7, P<0.05; ^& CSCs^Exo vs CSCs and control at postoperative day 28, P<0.05; ^# CSCs vs control at postoperative day 28, P<0.05.

Figure 10

MicroRNA (miRNA) expression profiling and validation of microarray data. A, miRNA profiles differentiate cardiac stem cells (CSCs) treated with mesenchymal stem cell (MSC) exosomes from nontreated CSCs (n=3). Both downregulated (green) and upregulated (red) miRNAs were identified in CSCs^Exo. B, Validation of microarray data using real‐time RT‐PCR. Triplicate assays were done for each RNA sample, and the relative amount of each miRNA was normalized to U6 snRNA.

Figure 11

Differentially expressed microRNAs (miRNAs) and gene ontology (GO) and pathway analysis based on miRNA‐targeted genes. A, List of the differentially expressed miRNAs and the fold‐changes are indicated. B, GOs targeted by overexpressed or underexpressed miRNAs. All these GOs show increased enrichment. The vertical axis is the GO category, and the horizontal axis is the enrichment of GO. C, Pathways targeted by overexpressed or downexpressed miRNAs. The vertical axis is the pathway category and the horizontal axis is the enrichment of pathways.

Figure 12

The microRNA (miRNA)–gene network. Blue and red box nodes represent miRNA, and purple cycle nodes represent mRNA. Edges describe the inhibitive effect of miRNA on mRNA. Blue box nodes show the underexpression microRNA–mRNA network, and red box nodes show the overexpression miRNA–mRNA network.