INDUCED PLURIPOTENT STEM CELL (iPSC)-DERIVED EXOSOMES FOR PRECISION MEDICINE IN HEART FAILURE

Heart failure and peri-infarct region (PIR) in ischemic cardiomyopathy patients

In the United States, heart failure (HF) is the leading cause of hospital admission and a major public health problem¹. Ischemic cardiomyopathy is the primary etiology of advanced HF¹. More than five million people suffer from HF, with an annual incidence of 300,000 patients¹. Despite significant therapeutic advances over the last three decades, the five-year survival remains at a dismal 50%². Clinical studies have confirmed that the high morbidity and mortality of HF are due to left ventricular (LV) arrhythmias and dilatation^1–2. Patients with a history of acute myocardial infarction (MI) and LV dysfunction have a six-month mortality of greater than 10% of which one-third is attributed to sudden cardiac death³. While implantable cardioverter defibrillators (ICDs) reduced mortality from LV arrhythmia, acute MI patients do not qualify under the current guideline. An important trigger for ventricular arrhythmias and dilatation is the tissue heterogeneity in the peri-infarct region (PIR), which is independent of the actual infarct size^4–7. An effective targeted therapy to salvage the injured and vulnerable myocardium in the PIR is urgently needed to reduce the high mortality of HF patients^7–8. Promising discoveries of iPSC biology may restore the PIR^9–10.

Salvage of the injured myocardium as a mechanism of stem cell therapy

The initial theory for the regeneration of the myocardium hypothesized that the transplanted stem cells would differentiate into cardiomyocytes, engraft into the host myocardium, and augment the cardiac function through synchronized electromechanical integration. However, de novo cardiac differentiation of transplanted stem cells was rare and mostly led to teratoma^9–10. Similarly, ex vivo differentiated cardiac stem cells demonstrate transient in vivo engraftment and functional restoration^9–10. Other controversial studies examined the cardiac progenitor cells, a multipotent population of cardiac, vascular, and endothelial cells in the myocardium. Both pre-clinical and clinical studies demonstrated significant restoration of cardiac function and viability following delivery; however, the immunohistology was equivocal regarding the functional role of these cells^11–12. A more physiologic explanation of the mechanism of action of these cells may be the salvage of the PIR by paracrine mechanism^8–10. Our work has demonstrated that the pluripotent stem cell-derivatives increase exosome production to enhance cytokine release and related gene expression to restore the viability of the PIR and ventricular function^8–10,13. Epicardial patch delivery of folistatin-1-like molecule activated endogenous paracrine mechanism, increased ventricular function, and enhanced myocardial viability¹³.

Exosomes provide a cell-free mechanism of action of iCMs to salvage the injured myocardium

Recent evidence indicates that the stem cells exert their therapeutic action via paracrine mechanisms through exosomes and show distinct therapeutic functionalities¹⁴. These vesicles are formed by invagination of the endosomal membrane with a diameter of 40 to 150 nm and released into the extracellular space. They contain unique cargo of cytoplasmic RNAs, molecules, and proteins to function as intercellular messengers and epigenetic regulators. Our data demonstrate that the exosomes and their cargo underlie the mechanism of action of iPSC-derived cardiomyocytes (iCMs) in salvaging the injured cardiomyocytes in the PIR against apoptosis, necrosis, inflammation, remodeling, and fibrosis. Endogenous paracrine secretion of the exosomes to rescue the surrounding PIR in response to ischemia is facilitated by specific transfer of RNAs, peptides, and small molecules. For an example, myocardial infarction activates the secretion of exosomes from the non-injured myocardium to reprogram the injured cardiomyocytes to trigger hypertrophy. These studies provide unique insights into the therapeutic potential of endogenous exosomes from the autologous iPSC-derivatives to translate precision medicine by personalizing cell-free clinical implementation of iPSC biology.

Exosomes selectively target the cell types of interest such as cardiomyocytes because exosomal membrane proteins and other components usually reflect their cellular origin and disease state¹⁴. The exosomal membrane can contain certain proteins that have binding affinities to specific receptors on the cell surface of the autologous cells and transfer naturally produced, therapeutic miRs for targeted, individualized therapy. For example, the exogenously introduced exosomes during myocardial injury may override the fibrotic response by the native cardiac fibroblasts by promoting myocardial repair through their anti-apoptotic, pro-angiogenic and proliferative effects, activating the specific pathways to mediate these pleiotropic effects to salvage the injured myocardium. No comparative study on the efficacy of the exosomes from different cell sources has been done. Thus, we can only speculate that the autologous exosomes allow patient-specific iCMs, containing endogenous injury-specific cargo, to target and self-repair the injured PIR.

Current State of the Art

Stem cell research stands at a critical juncture. Although the clinical potential of iCMs is obvious, no effective therapeutic strategy currently exists. Published data from our and other laboratories support the paracrine mechanism of these cells to salvage the PIR^8–13. This strategy is most physiologic by salvaging the injured myocardium while preserving the cytoarchitecture of the heart to rescue the injured cardiomyocytes in the PIR. Revascularization and/or ablation of arrhythmogenic tissue currently does not do this effectively. Our data demonstrate that the exosomes effectively salvage the injured myocardium. Our integrated workflow in the laboratory allows robust generation of iPSCs from patient’s serum, reliable differentiation into iCMs, and effective isolation and characterization of the exosomes¹⁴. Rigorous analysis of the salutary effects of the exosomes in pre-clinical murine and porcine myocardial injury models will enable rapid translation of exosome-based HF therapy. A continuous supply of the exosomes is generated from the iCMs. This framework will correlate the beneficial effects of the exosomes in murine and porcine myocardial injury models with their in vitro effects on the injured iCMs to delineate the mechanism of action. A logical and well-conceived pre-clinical studies will achieve FDA IND approval rapidly to conduct Phase I clinical trial of precision medicine, employing autologous iCM-derived exosomes in advanced HF patients.

The miRNA profile of the exosome cargo from the hypoxic iCMs was characterized and the mechanism of action to attenuate the injury of autologous hypoxia-injured iCMs was elucidated. Selective packaging of the miRNA cargo and their transfer occur by specific signaling molecules to promote self-healing of the injured heart¹⁵. This endogenous process allows disease-specific exosome expression for precision medicine. A specific set of miRNAs control the reparative processes and provide a compelling rationale for their therapeutic potential¹⁵. We identified a cluster of discrete group of adjacent miRNA genes on chromosome X. These miRNA clusters are transcribed as one pre-miRNA transcript and then cleaved into individual miRNAs¹⁵. They consist of similar sequences to regulate a set of mRNAs, which control a defined set of cellular activities. They are transcribed by a common promoter and express a high sequence homology to target a unique set of mRNAs within a same pathway. We identified the miR-106a-363 cluster, consisting of 6 miRNAs (miR-106a, -18b, 19b, -20b, -92a, -363). This group of miRs is one of the paralogues of the miR-17–92 cluster and a well-conserved miRNA cluster in both human and murine genome^14–15. This cluster has been reported mostly in the cancer field and never investigated in human cardiovascular disease. In our preliminary study, we investigated the role of the miR-106a-363 cluster carried by the exosomes from the hypoxia injured iCMs. This miRNA cluster was consistently enriched in the exosomes secreted from the hypoxia injured iCMs compared to the exosomes from the normoxia healthy (control) iCMs. Furthermore, our findings revealed that the JAG1-NOTCH3-HES1 axis as a direct target pathway of the miRNA-106a-363 cluster, which activate anti-apoptotic and -fibrotic effects to preserve the cardiac function. These results indicate that the endogenous production of the exosomal miRNA-106a-363 cluster activates the homeostatic self-repair of the ischemia-injured myocardium by targeting the NOTCH3 gene and related signaling pathway.

Conclusion

The ability to generate individualized iPSCs from a single blood draw and differentiate them readily into iCMs offers an exceptional translational promise through a continuous supply of autologous exosomes. Our optimized iCM platform generates >90% pure population of contractile cells and produces unlimited supply of exosomes in response to ischemic stress to study their restorative effects on the autologous hypoxia-injured iCMs. Administration of the exosomes generated from the hypoxic iCMs back to the hypoxia injured iCMs confirmed their therapeutic role in endogenous repair. Furthermore, significant in vivo functional benefit in both murine and porcine myocardial injury model was shown. Our molecular analyses of the therapeutic effects of the exosomes on the in vitro iCMs correlated with the changes on ex vivo murine and porcine myocardium to elucidate the mechanism of action of in vivo endogenous salvage.

Our approach represents a potential paradigm shift in stem cell therapy through the investigation of the feasibility of patient- and disease-specific exosomes for the repair of the injured myocardium. Our multi-disciplinary study demonstrated the fundamental insights into novel mechanisms of action. The autologous exosomes are injected directly into the PIR of the injured myocardium without any risk for suboptimal cell delivery, survival and engraftment. This precision medicine approach employs the exosomes from the autologous iCMs to trigger endogenous repair through disease-specific exposure to hypoxia, simulating myocardial ischemia. The resultant exosomes offer the potential for highest therapeutic efficacy due to their cell free, autologous, and disease-specific approach to HF. More importantly, the pluripotency of iPSCs will enable exosome production from any human tissue cell and allow a wide range of treatment for degenerative diseases (Figure).

Figure. Exosome therapy.

Following peripheral blood draw, mononuclear cells are reprogrammed by Sendai virus to generate patient-specific iPSCs. The iPSCs undergo robust cardiac differentiation using small molecules. Exosomes are readily isolated from the iCM supernatant. The specific advantages of exosome are illustrated. This scheme applies to iPSC-derivatives differentiated into any cell lineage.