Mining Exosomal MicroRNAs from Human-Induced Pluripotent Stem Cells-Derived Cardiomyocytes for Cardiac Regeneration

Cardiovascular diseases (CVD) are the primary cause of morbidity and mortality worldwide [1]. Following myocardial infarction, there is a major loss of cardiomyocytes due to necrosis, and replacement of damaged myocardium is an intense area of research in the field of cardiac regenerative medicine [2]. Various cell types, including mesenchymal stem cells (MSCs), cardiac progenitor cells (CPCs), embryonic stem cells-derived cardiomyocytes (ESC-CMs), and induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs), have been investigated in both preclinical and clinical studies with mixed results, with a common issue being the poor survival of transplanted cells [3–7]. Positive outcomes of such studies are been associated with the secretion of paracrine factors in the absence of adequate cell survival, which may be mediated by the release of a novel class of small vesicles termed exosomes [7]. Exosomes are endosomal membrane vesicles with diameters of ~40–150 nm that originate in the late endosomal compartment from the inward budding of endosomal membranes, leading to the formation of intracellular multi-vesicular endosomes (MVEs) [8,9]. Groups of exosomes are packed in the MVEs and upon their fusion with the plasma membrane, these exosomes are released into the extracellular space. Various studies have reported the role of exosomes in mediating intracellular communication by serving as vehicles for transferring various biological molecules, including DNA, RNA, proteins, and lipids between cells [10–13]. Consistent with these studies, the roles of exosomes isolated from various types of stem cells such as MSCs, CPCs, ESCs, iPSCs and CD34+ have been investigated in mediating cardiac regeneration. These exosomes are known to possess anti-apoptotic effects in both host as well as transplanted cells, and they also stimulate angiogenesis, reduce infarct size, and improve cardiac recovery [14–18]. Importantly, enrichment of protective microRNAs appears to be responsible for the beneficial effects of exosomes in cardiac regeneration [14,10,19,20]. In the following text, we have outlined a protocol for differentiating human iPSCs into cardiomyocytes, and described the methods required for isolating exosomes secreted by iPSC-CMs for subsequent microRNA analysis, which will help advance our understanding of exosomes and how they can be used for mediating cardiac regeneration moving forward.