Ultrastructural evidence of exosome secretion by progenitor cells in adult mouse myocardium and adult human cardiospheres

Abstract

The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres) that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34⁺ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an "off-the-shelf" product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

Figure 1

Left and middle panels: Flow cytometric analysis of the cellular outgrowth from a human atrial appendage explant in the primary tissue culture, showing MSC (CD13⁺, CD73⁺, and CD105⁺) but not hematopoietic (CD45⁻) marker expression. Upper right panel: Photomicrograph of a human atrial explant with cellular outgrowth; middle right panel: human CSs; lower right panel: human CDCs.

Figure 2

Immunostaining of a human CS showing expression of cardiac troponin I (red); nuclei stain blue.

Figure 3

Transmission electron micrographs showing a cardiac progenitor cell (CPC), characterized by a large nucleus and a thin cytoplasmic rim, in adult mouse myocardium (a) and in an adult human CS in vitro (b).

Figure 4

Transmission electron micrographs showing exosomes (≈75 nm diameter; Exo. (a)) and microvesicles (≈200 nm diameter; (b)) secreted by cardiac progenitor cells (CPC) in a human CS in vitro. Exosomes in adult mouse myocardium ((c)-(d); TC: telocyte; ESC: extracellular space).

Figure 5

Transmission electron micrographs showing exosomes in an adult mouse heart. (a) Exosomes (asterisks) located at the cell surface of a cardiomyocyte (CM). The latter extends thin cytoplasmic processes (arrow) that encircle the exosome. Inset: higher magnification view showing dense nanostructures (arrowheads) at the interface of the CM membrane and the membrane of the exosome, suggesting involvement of molecular interactions in exosome uptake by CMs. (b) Exosomes (asterisks) encircled by cytoplasmic processes (arrow). (c) Exosomes (asterisks) taken up by the CM within small, round cytoplasmic structures (arrows).