Human Cardiac Progenitor Cell-Derived Extracellular Vesicles Exhibit Promising Potential for Supporting Cardiac Repair in Vitro

Abstract

Although human Cardiac Progenitor Cells (hCPCs) are not retained by host myocardium they still improve cardiac function when injected into ischemic heart. Emerging evidence supports the hypothesis that hCPC beneficial effects are induced by paracrine action on resident cells. Extracellular vesicles (EVs) are an intriguing mechanism of cell communication based on the transport and transfer of peptides, lipids, and nucleic acids that have the potential to modulate signaling pathways, cell growth, migration, and proliferation of recipient cells. We hypothesize that EVs are involved in the paracrine effects elicited by hCPCs and held accountable for the response of the infarcted myocardium to hCPC-based cell therapy. To test this theory, we collected EVs released by hCPCs isolated from healthy myocardium and evaluated the effects they elicited when administered to resident hCPC and cardiac fibroblasts (CFs) isolated from patients with post-ischemic end-stage heart failure. Evidence emerging from our study indicated that hCPC-derived EVs impacted upon proliferation and survival of hCPCs residing in the ischemic heart and regulated the synthesis and deposition of extracellular-matrix by CFs. These findings suggest that beneficial effects exerted by hCPC injection are, at least to some extent, ascribable to the delivery of signals conveyed by EVs.

Keywords: cardiac regenerative medicine; extracellular vesicles; human cardiac fibroblasts; human cardiac progenitor cells; paracrine effects.

FIGURE 1

Experimental design. Schematic representation of the study.

FIGURE 2

Comparative analysis and quantification of growth factor content in EV-CPC-N and EV-CPC-P cargos. Array map is used as a reference (A) and on protein array membranes are highlighted higher amounts of EGF, FGF, IGF-1, and SCF (D) in EV-CPC-N (B) or higher amounts of HGF, SCF-R, TGF, VEGF, and its receptors (D) in EV-CPC-P (C).

FIGURE 3

Gene expression analysis of cardiac cell markers in EV-CPC-N and EV-CPC-P cargos. Real-time PCR analysis of the gene expression for markers characteristic of cardiac myocytes, smooth muscle cells and mesenchymal cells showed a downregulated transcription of the cardiac myocyte markers MEF2C and NKX2.5, and an upregulated transcription of the smooth muscle cell marker ACTA2 in EV-CPC-P. (*p ≤ 0.05 vs. EV-CPC-N).

FIGURE 4

Analysis of α-sarcomeric actin expression at gene or protein level of CPC-P cultured in the absence or in the presence of EV-CPC-N. Graphical representation of ACTC1 expression by Real-time PCR (A) and quantification of the α-sarcomeric actin immunopositivity (B) in CPC-P cultured with EV-CPC-N vs. CPC-P cultured without EV-CPC-N (*p ≤ 0.05 vs. CPC-P). Analysis of the morphology and α-sarcomeric actin expression of CPC-P cultured in the absence or in the presence of EV-CPC-N. (C) and (D) Representative images acquired at the phase contrast microscope showing a similar morphology between CPC-P cultured without (C) or with (D) EV-CPC-N. E and (F) Representative images of immunofluorescence analysis showing by the green fluorescence the α-sarcomeric actin expression and distribution pattern in CPC-P cultured without (E) or with (F) EV-CPC-N. The blue fluorescence is the result of nuclear counterstaining with DAPI. Scale bar: 250 μm for (C) and (D), and 200 μm for (E) and (F).

FIGURE 5

Evaluation of the proliferation and apoptotic rate of CPC-P cultured in the absence or in the presence of EV-CPC-N. (A–C): Representative light microscopy images of the live CPC-P (A) and CPC-P cultured with EV-CPC-N (B) detection in the G1 (yellow), G2-S (green) or M (blue) phases of the cell cycle and their quantification (C) using the Cell-Clock Cell Cycle Assay. (D–F): Representative light microscopy images of the detection of CPC-P apoptosis (purple-red colored cells) as related to the absence (D) or the presence (E) of EV-CPC-N and quantification of apoptotic rate (F) using the Cell-APOPercentage Apoptosis Assay. Asterisks are indicators of the p value as follows: significant (*p ≤ 0.05 vs. CPC-P) and very significant (**p ≤ 0.01 vs. CPC-P). Scale bar: 100 μm.

FIGURE 6

Evaluation of the speed of migration of CPC-P cultured in the absence or in the presence of EV-CPC-N. The scratch wound assay allowed the measurement of cell migration speed. Migration was not affected by administration of EV-CPC-N (A) and complete closure of scratch wound made in CPC-P adherent monolayer cultured in the absence (B) or in the presence (C) of EV-CPC-N occurred within 8 h [(D) and (E), respectively]. Scale bar: 200 μm.

FIGURE 7

Gene expression analysis of specific transcription of ECM proteins in CF-P cultured in absence or in the presence of EV-CPC-N. Real-time PCR analysis showed a significant up-regulation of genes encoding for collagen type IV and fibronectin. No statistically significant differences emerged for laminin, tenascin, collagen type I and type III (*p ≤ 0.05 vs. CF-P).

FIGURE 8

Microscopic analysis of the morphology and immunopositivity for fibronectin of CF-P cultured in the absence or in the presence of EV-CPC-N. (A) and (B) representative images acquired at phase contrast microscope showing the morphology of CF-P cultured in the absence (A) or in the presence (B) of EV-CPC-N. (C) and (D) Representative images of immunofluorescence analysis showing by the green fluorescence the fibronectin distribution pattern in CF-P cultured in the absence (C) or in the presence (D) of EV-CPC-N. The blue fluorescence is the result of nuclear counterstaining with DAPI. Scale bar: 200 μm for A and B, 250 μm for (C) and (D).