Induced Pluripotent Stem Cells Derived Extracellular Vesicles: A Potential Therapy for Cardiac Repair

“The science of today is the technology of tomorrow”.

Edward Teller

Cardiovascular disease (CVD) remains one of the major health issues in the US. Existing therapies prolong the life of the patients but do not actually regenerate the lost cardiac muscle tissue. In this perspective, evidence from animal models of experimental ischemic injuries suggest that exogenous transfer of a variety of stem/progenitor cells orchestrate functional improvement and ischemic tissue repair/regeneration after cardiac injury¹. One of the primary features of stem cells relates to their ability to differentiate into different types of cells and tissues. This potential to ‘regenerate’ tissues was the primary rationale behind the early studies of stem cell therapy in animal models, and subsequently in humans. However, with the rapid increase in studies that show very little or no replacement of lost cells through trans-differentiation of stem cells, the support for the ‘paracrine effects’ theory has grown. It is now generally believed that adult stem cells induce organ repair primarily via mechanisms other than differentiating into another cell type, such as a cardiomyocyte.

Pluripotent embryonic stem cells (ESCs) have shown a great promise in cardiac regeneration, due to their unparalleled cardiomyocyte differentiation potential². However, major obstacles using embryonic stem cells transplantation includes immune rejection, arrhythmogenesis, and tumorigenic potential³. The promise of true regeneration of damaged or lost tissues was greatly enhanced in 2006 with the discovery of induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka that showed the traits of a truly pluripotent cells and revolutionized the field of regenerative medicine as somatic cells can now be reprogrammed to pluripotent stem cells⁴, thus circumventing the immune rejection and ethical issues associated with ESCs⁵. The production of these cells from humans was hailed widely as the beginning of an era wherein one could ‘reprogram’ a patient’s own cells and then differentiate down a desired pathway to repair his or her own organs. Although this sounds almost too good to be true, abundant scientific evidence in elite journals supports this possibility. However, similar to ESC, iPSCs also induce tumor formation⁶ and like other cellular therapies, iPSCs might also suffer issues such as cell viability, retention and engraftment.

Another major discovery in the field of regenerative medicine in recent years was the reincarnation of extracellular vesicles (EVs), small membrane-enclosed droplets that carry biologically active molecules and genetic materials from parent cells. Emerging evidence suggests that stem/progenitor cell derived EVs provide an alternate cell-free therapeutic modality for cardiac repair. Novel, non-traditional use of cell-free components of stem/progenitor cells such as EVs, loaded with stem cell-specific RNAs and proteins may allow harnessing the regenerative power of these cells to augment and modulate endogenous protection by transferring the cargo to various cardiovascular cells and stimulate repair processes in the ischemic myocardium^{7, 8}.

So which is better? iPSCs that can ‘regenerate’ the lost tissue, or EVs that can repair the damaged organ through paracrine actions without necessarily producing new cells? This is a difficult question for which there is no easy answer. In this issue of Circulation Research Adamiak et. al.⁹ report data from the first head-to-head comparison of cardiac reparative effects of iPSCs and iPSC-derived EVs that may help resolve this issue. Their results show that improvement in left ventricular (LV) function was better with EVs, although iPSCs also improved function. Remodeling was similar in both groups except for lower LV mass with EV therapy. Interestingly, the reduction in apoptosis and vascularity in the infarct zone were significantly better EV therapy. The authors also performed thorough characterization of the contents of iPSC-derived EVs, which showed the presence of several miRNAs and proteins in EV cargo that influence cell survival among many other important biological functions. So, both forms of therapies were beneficial, with EVs coming out somewhat better compared with parental cells. The authors went on to show that over 50% mice injected with iPSCs developed teratomas but none of the mice injected with iPSC-EVs developed teratomas, even though these EVs were derived from these iPSCs that caused tumors. This is a an important finding that underscores the importance of safety vis-à-vis efficacy of any therapy considered for human applications. Of note, authors cultured iPSCs in feeder-free and serum-free conditions to exclude the contaminant RNA/lipoproteins usually co-isolated with the EVs, which can influence EVs biological effects on recipient cells. Thus, as demonstrated in this study, reproducible stem/ progenitor EVs isolation using serum-free conditions may have potential future clinical applications to accelerate cardiac repair.

iPSCs are indeed able to differentiate into cells of diverse lineages, consistent with their name. But is this a good thing or a bad thing for therapeutic regeneration? A hallmark of bona fide iPSCs is their ability to form teratomas in vivo. If this tendency to differentiate can be curbed appropriately so that the modified iPSCs are only able to form one type of cells, then of course the pluripotent nature is a good thing – to produce cells for many different organs, if necessary. Several reports indicate that iPSCs can be modified in culture to make cardiomyocytes only, and the resultant myocytes can be used for heart repair. On the contrary, if this lineage-directed specification is somehow not absolutely 100% successful for every cell in an inoculum of millions of cells, then perhaps iPSCs can cause collateral damage that can be even fatal for the patient in the worst case scenario. Irrespective of the candidacy of iPSCs for cardiac repair in patients, it is reassuring to know that EVs from iPSCs also induce infarct repair with outcomes that are better than or at least comparable to those seen with iPSCs. Preservation of the viable myocardium in the infarct zone was similar with these two types of treatments, and both better than the control group. This is encouraging, because more myocytes in the scar region is a good thing for cardiac contractility, even though the underlying mechanism for EVs may be different. It should be noted that the function and content of exosomes is dependent upon the parental cell; the therapeutic efficacy of iPSC-EVs may reflect the naïve undifferentiated pluripotent status of the iPSC. It is conceivable that directed differentiation of iPSCs to cardiomyocyte may alter the functional properties of the EVs derived from the differentiated cells.

The variability of different kinds of vesicles within heterogeneous EV population creates a challenge for distinguishing the functional vesicle type within all the EVs secreted by a cell. The EVs used in this study appear to be heterogeneous comprising of both exosomes and ectosomes. Unlike exosomes, ectosomes or micro vesicles are a sub-class of EVs which their diameters ranging 0.1–1µm. These vesicles which bud directly from the plasma membrane, typically carry cell surface markers and cytosolic contents similar to parental cell¹⁰. Unlike, exosomes, the molecular composition of ectosomes is not well established. Future studies on potential differences in terms of exosomes/ectosomes cargo and therapeutic efficacy of each fraction of EVs separately remains to be investigated. Of note, a recent study on quantitative and stoichiometric analysis of the miRNA content of exosomes demonstrated that over 100 exosomes would be required to fuse to a recipient cell to transfer a single copy of a highly abundant endogenous miRNA, suggesting a significant amount of miRNA should be transferred to have a biological effect¹¹. Therefore, it would be of interesting to investigate if certain miRNA/s in iPSC-EVs are abundant and more important than others? Further, a recent study demonstrated that iPSC-cardiomyocytes derived exosomes carry miRNAs and lncRNAs¹². Although, data from current study supports the role of miRNAs and proteins enriched in iPSC-EVs that augment cardiac repair post- myocardial repair, future studies are warranted to perform deep sequencing analysis to comprehend the role of other non-coding RNAs in mediating EVs functional benefits observed here. Also, given that iPSC-EVs carry miRNAs and each miRNA has multiple target genes, future efforts should also focus on EVs specifically targeting cardiac tissue to prevent any adverse off-target effects. Despite these lingering questions, the study by Adamiak et. al.⁹ provides a valuable information to the active field of cardiac repair and regeneration by establishing both safety and efficacy of iPSC-EVs for cardiac repair. Future studies focused on delineating a more comprehensive mechanistic insights are likely to establish that iPSCs-EV therapy could potentially be applicable for future clinical applications in cardiovascular regenerative medicine.